Methods and compositions for treating diabetes, metabolic syndrome and other conditions

ABSTRACT

Pharmaceuticals compositions comprising the 2S,4R, ketoconazole enantiomer or its pharmaceutically acceptable salts, hydrates, and solvates, that are substantially free of the 2R,4S ketoconazole enantiomer are useful to reduce cortisol synthesis and for the treatment of type 2 diabetes, hyperglycemia, obesity, insulin resistance, dyslipidemia, hyperlipidemia, hypertension, Metabolic Syndrome, and other diseases and conditions, including but not limited to Cushing&#39;s Syndrome, depression, and glaucoma.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.16/715,690, filed Dec. 16, 2019, now U.S. Pat. No. 10,835,530, which isa continuation of U.S. application Ser. No. 16/152,202, filed Oct. 4,2018, now U.S. Pat. No. 10,517,868, which is a continuation of U.S.application Ser. No. 15/886,437, filed Feb. 1, 2018, now U.S. Pat. No.10,098,877, which is a continuation of U.S. application Ser. No.15/088,539, filed Apr. 1, 2016, now U.S. Pat. No. 9,918,984, which is acontinuation of U.S. application Ser. No. 11/813,662, filed May 21,2008, now abandoned, which is the United States national stage ofInternational Application No. PCT/IB2006/000026, filed Jan. 10, 2006,which claims benefit of U.S. Provisional Application No. 60/643,055,filed Jan. 10, 2005, all of which are herein incorporated by referencein their entirety.

FIELD OF THE INVENTION

The present invention relates to pharmaceutical compositions and methodsfor treating diabetes and other conditions, including type 2 diabetesmellitus, metabolic syndrome, insulin resistance, obesity, lipiddisorders, metabolic disease, and other conditions that can be treatedby reducing cortisol synthesis, including but not limited to Cushing'sSyndrome, osteoporosis, glaucoma and depression. The invention thereforerelates to the fields of chemistry, biology, pharmacology, and medicine.

BACKGROUND OF THE INVENTION

Ketoconazole,1-acetyl-4-[4-[[2-(2,4-dichlorophenyl)-2-[(1H-imidazol-1-yl)-methyl]-1,3-dioxolan-4-yl]methoxy]phenyl]piperazine,is a racemic mixture of the cis enantiomers (−)-(2S,4R) and (+)-(2R,4S)marketed as an anti-fungal agent. Ketoconazole inhibits fungal growththrough the inhibition of ergosterol synthesis. Ergosterol is a keycomponent of fungal cell walls.

More recently, ketoconazole was found to decrease plasma cortisol and tobe useful, alone and in combination with other agents, in the treatmentof a variety of diseases and conditions, including type 2 diabetes,Metabolic Syndrome (also known as the Insulin Resistance Syndrome,Dysmetabolic Syndrome or Syndrome X), and other medical conditions thatare associated with elevated cortisol levels. See U.S. Pat. Nos.5,584,790; 6,166,017; and 6,642,236, each of which is incorporatedherein by reference. Cortisol is a stress-related hormone secreted fromthe cortex of the adrenal glands. ACTH (adenocorticotropic hormone)increases cortisol secretion. ACTH is secreted by the pituitary gland, aprocess activated by secretion of corticotropin releasing hormone (CRH)from the hypothalamus.

Cortisol circulates in the bloodstream and activates specificintracellular receptors, such as the glucocorticoid receptor (GR).Disturbances in cortisol levels, synthetic rates or activity have beenshown to be associated with numerous metabolic complications, includinginsulin resistance, obesity, diabetes and Metabolic Syndrome.Additionally, these metabolic abnormalities are associated withsubstantially increased risk of cardiovascular disease, a major cause ofdeath in industrialized countries. See Mårin P et al., “Cortisolsecretion in relation to body fat distribution in obese premenopausalwomen.” Metabolism 1992; 41:882-886, Bjomtorp, “Neuroendocrineperturbations as a cause of insulin resistance.” Diabetes Metab Res Rev1999; 15(6): 427-41, and Rosmond, “Role of stress in the pathogenesis ofthe metabolic syndrome.” Psychoneuroendocrinology 2005; 30(1): 1-10,each of which is incorporated herein by reference.

While ketoconazole is known to inhibit some of the enzymatic steps incortisol synthesis, such as, for example, 17α hydroxylase (Wachall etal., “Imidazole substituted biphenyls: a new class of highly potent andin vivo active inhibitors of P450 17 as potential therapeutics fortreatment of prostate cancer.” Bioorg Med Chem 1999; 7(9): 1913-24,incorporated herein by reference) and 11b-hydroxylase (Rotstein et al.,“Stereoisomers of ketoconazole: preparation and biological activity.” JMed Chem 1992; 35(15): 2818-25) and 11β-hydroxy steroid dehydrogenase(11β-HSD) (Diederich et al., “In the search for specific inhibitors ofhuman 11β-hydroxysteroid-dehydrogenases (11β-HSDs): chenodeoxycholicacid selectively inhibits 11β-HSD-I.” Eur J Endocrinol 2000; 142(2):200-7, incorporated herein by reference) the mechanisms by whichketoconazole decreases cortisol levels in the plasma have not beenreported. For example, there is uncertainty regarding the effect ofketoconazole on the 11β-hydroxy steroid dehydrogenase (11β-HSD) enzymes.There are two 11β-HSD enzymes. One of these, 11β-HSD-I, is primarily areductase that is highly expressed in the liver and can convert theinactive 11-keto glucocorticoid to the active glucocorticoid (cortisolin humans and corticosterone in rats). In contrast, the other,11β-HSD-II, is primarily expressed in the kidney and acts primarily asan oxidase that converts active glucocorticoid (cortisol in humans andcorticosterone in rats) to inactive 11-keto glucocorticoids. Thus, theplasma concentration of active glucocorticoid is influenced by the rateof synthesis, controlled in part by the activity of adrenal11β-hydroxylase and by the rate of interconversion, controlled in partby the relative activities of the two 11β-HSD enzymes. Ketoconazole isknown to inhibit these three enzymes (Diederich et al., supra) and the2S,4R enantiomer is more active against the adrenal 11β-hydroxylaseenzyme than is the 2R,4S enantiomer (Rotstein et al., supra). However,there are no reports describing the effect of the two ketoconazoleenantiomers on either of 11β-HSD-I or 11β-HSD-II, so it is not possibleto predict what effects, if any, the two different ketoconazoleenantiomers will each have on plasma levels of the active glucocorticoidlevels in a mammal.

Ketoconazole has also been reported to lower cholesterol levels inhumans (Sonino et al. (1991). “Ketoconazole treatment in Cushing'ssyndrome: experience in 34 patients.” Clin Endocrinol (Oxf). 35(4):347-52; Gylling et al. (1993). “Effects of ketoconazole on cholesterolprecursors and low density lipoprotein kinetics inhypercholesterolemia.” J Lipid Res. 34(1): 59-67) each of which isincorporated herein by reference). The 2S,4R enantiomer is more activeagainst the cholesterol synthetic enzyme 14αlanosterol demethylase thanis the other (2R,4S) enantiomer (Rotstein et al infra). However, becausecholesterol level in a human patient is controlled by the rate ofmetabolism and excretion as well as by the rate of synthesis it is notpossible to predict from this whether the 2S,4R enantiomer ofketoconazole will be more effective at lowering cholesterol levels.

The use of ketoconazole as a therapeutic is complicated by the effect ofketoconazole on the P450 enzymes responsible for drug metabolism.Several of these P450 enzymes are inhibited by ketoconazole (Rotstein etal., supra). This inhibition leads to an alteration in the clearance ofketoconazole itself (Brass et al., “Disposition of ketoconazole, an oralantifungal, in humans.” Antimicrob Agents Chemother 1982; 21(1): 151-8,incorporated herein by reference) and several other important drugs suchas Glivec (Dutreix et al., “Pharmacokinetic interaction betweenketoconazole and imatinib mesylate (Glivec) in healthy subjects.” CancerChemother Pharmacol 2004; 54(4): 290-4) and methylprednisolone (Glynn etal., “Effects of ketoconazole on methylprednisolone pharmacokinetics andcortisol secretion.” Clin Pharmacol Ther 1986; 39(6): 654-9). As aresult, the exposure of a patient to ketoconazole increases withrepeated dosing, despite no increase in the amount of drug administeredto the patient. This exposure and increase in exposure can be measuredand demonstrated using the “Area under the Curve” (AUC) or the productof the concentration of the drug found in the plasma and the time periodover which the measurements are made. The AUC for ketoconazole followingthe first exposure is significantly less than the AUC for ketoconazoleafter repeated exposures. This increase in drug exposure means that itis difficult to provide an accurate and consistent dose of the drug to apatient. Further, the increase in drug exposure increases the likelihoodof adverse side effects associated with ketoconazole use.

Rotstein et al. (Rotstein et al., supra) have examined the effects ofthe two ketoconazole cis enantiomers on the principal P450 enzymesresponsible for drug metabolism and reported “ . . . almost noselectivity was observed for the ketoconazole isomers” and, referring todrug metabolizing P450 enzymes: “[t]he IC50 values for the cisenantiomers were similar to those previously reported for racemicketoconazole”. This report indicated that both of the cis enantiomerscould contribute significantly to the AUC problem observed with theketoconazole racemate.

One of the adverse side effects of ketoconazole administrationexacerbated by this AUC problem is liver reactions. Asymptomatic liverreactions can be measured by an increase in the level of liver specificenzymes found in the serum and an increase in these enzymes has beennoted in ketoconazole treated patients (Sohn, “Evaluation ofketoconazole.” Clin Pharm 1982; 1(3): 217-24, and Janssen and Symoens,“Hepatic reactions during ketoconazole treatment.” Am J Med 1983;74(1B): 80-5, each of which is incorporated herein by reference). Inaddition 1:12,000 patients will have more severe liver failure (Smithand Henry, “Ketoconazole: an orally effective antifungal agent.Mechanism of action, pharmacology, clinical efficacy and adverseeffects.” Pharmacotherapy 1984; 4(4): 199-204, incorporated herein byreference). As noted above, the amount of ketoconazole that a patient isexposed to increases with repeated dosing even though the amount of drugtaken per day does not increase (the “AUC problem”). The AUC correlateswith liver damage in rabbits (Ma et al., “Hepatotoxicity andtoxicokinetics of ketoconazole in rabbits.” Acta Pharmacol Sin 2003;24(8): 778-782 incorporated herein by reference) and increased exposureto the drug is believed to increase the frequency of liver damagereported in ketoconazole treated patients.

Additionally, U.S. Pat. No. 6,040,307, incorporated herein by reference,reports that the 2S,4R enantiomer is efficacious in treating fungalinfections. This same patent application also reports studies onisolated guinea pig hearts that show that the administration of racemicketoconazole may be associated with an increased risk of cardiacarrhythmia, but provides no data in support of that assertion. However,as disclosed in that patent, arrhythmia had not been previously reportedas a side effect of systemic racemic ketoconazole, although a particularsubtype of arrhythmia, torsades de pointes, has been reported whenracemic ketoconazole was administered concurrently with terfenadine.Furthermore several published reports (for example, Morganroth et al.(1997). “Lack of effect of azelastine and ketoconazole coadministrationon electrocardiographic parameters in healthy volunteers.” J ClinPharmacol. 37(11): 1065-72) have demonstrated that ketoconazole does notincrease the QTc interval. This interval is used as a surrogate markerto determine whether drugs have the potential for inducing arrhythmia.U.S. Pat. No. 6,040,307 also makes reference to diminished hepatoxicityassociated with the 2S,4R enantiomer but provides no data in support ofthat assertion. The method provided in U.S. Pat. No. 6,040,307 does notallow for the assessment of hepatoxicity as the method uses microsomesisolated from frozen tissue.

Thus, there remains a need for new therapeutic agents and methods fortreating diseases and conditions associated with elevated cortisollevels or activity or that may be treated by lowering cortisol level oractivity that are as effective as ketoconazole but do not present, orpresent to a lesser degree, the issues of drug interactions and adverseside effects of ketoconazole. The present invention meets these andother needs.

SUMMARY OF THE INVENTION

The present invention arises in part from the discoveries that the 2S,4Renantiomer is more effective per weight unit than racemic ketoconazoleor the 2R,4S enantiomer (the other enantiomer in the racemate) atreducing the concentration of the active glucocorticoid in the plasmaand that the 2S,4R enantiomer does not lead to drug accumulation (oraccumulates to a significantly less extent) as does racemicketoconazole.

In a first aspect, the present invention provides methods for treatingdiseases and conditions associated with elevated cortisol levels,production rates or activity and other diseases and conditions that canbe treated by reducing cortisol, or diseases or conditions that can betreated by reducing cholesterol levels, production rates or activity byadministering a pharmaceutical composition containing a therapeuticallyeffective amount of the 2S,4R ketoconazole enantiomer substantially orentirely free of the 2R,4S ketoconazole enantiomer.

In a second aspect, the present invention provides pharmaceuticalcompositions comprising a pharmaceutically acceptable carrier and atherapeutically effective amount of the 2S,4R ketoconazole enantiomersubstantially or entirely free of the 2R,4S ketoconazole enantiomerformulated for use in the methods of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the effect of the four ketoconazole enantiomers2S,4S,2R,4R,2R,4S, and 2S,4R on plasma corticosterone. The figure showsthat the 2S,4R enantiomer is more effective at lowering corticosteronethan any of the other three enantiomers. The concentration ofcorticosterone in the plasma of Sprague-Dawley rats was determined fourhours after delivery by oral gavage of 200 mg/kg of the indicatedenantiomer.

FIG. 2 shows the effect of racemic ketoconazole and of the two cisenantiomers 2R,4S and 2S,4R on plasma corticosterone. The 2S,4Renantiomer is more effective at lowering corticosterone than eitherracemic ketoconazole or the other enantiomer present in racemicketoconazole (2R,4S). The concentration of corticosterone in the plasmaof Sprague-Dawley rats was determined four hours after delivery by oralgavage of the indicated amount of either racemic ketoconazole or the twoenantiomers (2S,4R and 2R,4S) present in racemic ketoconazole.

FIG. 3 shows the effect of racemic ketoconazole or the two enantiomers2R,4S and 2S,4R on the time course of depression of plasmacorticosterone. The 2S,4R enantiomer is more effective at loweringcorticosterone than either racemic ketoconazole or the other cisenantiomer present in racemic ketoconazole (2R,4S). The concentration ofcorticosterone in the plasma of Sprague-Dawley rats was determined atthe indicated time after delivery by oral gavage of 200 mg/kg of eitherracemic ketoconazole or the two enantiomers (2S,4R and 2R,4S) present inracemic ketoconazole.

FIG. 4 shows the effect of prior exposure to ketoconazole on thepharmacokinetic profile of racemic ketoconazole in dogs. Thepharmacokinetic profile of racemic ketoconazole is clearly altered byprior exposure to racemic ketoconazole. The concentration of racemicketoconazole in the plasma of dogs that were dosed with racemicketoconazole daily for 28 days (in two different forms: in suspension inolive oil and in a solid tablet form) is significantly greater than theconcentration of racemic ketoconazole in the plasma of dogs that weretreated only once.

FIG. 5 shows the effect of prior exposure to racemic ketoconazole on thepharmacokinetic profile of racemic ketoconazole in dogs. The Area Underthe Curve (AUC) of racemic ketoconazole is increased by prior exposureto racemic ketoconazole. The AUC of the pharmacokinetic profile shown inFIG. 4 was calculated according to the trapezoid rule. The AUC ofracemic ketoconazole is greater in dogs treated daily for 28 days ascompared to dogs treated only once. The increase in AUC is independentof the form in which the racemic ketoconazole was administered.

FIG. 6 shows the effect of prior exposure to the 2S,4R enantiomer ofketoconazole on the pharmacokinetic profile of the 2S,4R enantiomer ofketoconazole in dogs. The pharmacokinetic profile of the 2S,4Renantiomer of ketoconazole is not altered by prior exposure to the 2S,4Renantiomer of ketoconazole. The concentration of the 2S,4R enantiomer ofketoconazole in the plasma of dogs that were dosed either once with the2S,4R enantiomer or were dosed daily for 28 days is not increased in thedogs treated for 28 days as compared to dogs treated only once.

FIG. 7 shows the effect of prior exposure to the 2S,4R enantiomer ofketoconazole on the AUC of the 2S,4R enantiomer of ketoconazole in dogs.The AUC of 2S,4R enantiomer of ketoconazole is not increased by priorexposure to the 2S,4R enantiomer of ketoconazole. The AUC of the 2S,4Renantiomer of ketoconazole is the same in dogs treated daily for 28 daysas compared to dogs treated only once.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides pharmaceutical compositions comprisingthe 2S,4R ketoconazole enantiomer substantially or entirely free of the2R,4S enantiomer, and methods of using such compositions. Substantiallyfree of the 2R,4S enantiomer, in one embodiment, means that theketoconazole content of the pharmaceutical composition is less than 2%of the 2R,4S enantiomer and more than 98% of the 2S,4R enantiomer. Inanother embodiment, substantially free of the 2R,4S enantiomer means theketoconazole content of the pharmaceutical composition is less than 10%of the 2R,4S enantiomer and more than 90% of the 2S,4R enantiomer. Inanother embodiment, substantially free of the 2R,4S enantiomer meansthat the ketoconazole content of the pharmaceutical composition is lessthan 20% of the 2R,4S enantiomer and more than 80% of the 2S,4Renantiomer. The present invention also provides methods for treatingdiseases and conditions associated with elevated cortisol levels oractivity and diseases and conditions that may be medically treated byreducing cortisol levels and cortisol activity with these pharmaceuticalcompositions. To aid in understanding the invention, this detaileddescription is organized as follows. Section I describes methods forpreparing the 2S,4R enantiomer, its solvates and salts, andpharmaceutical compositions comprising it. Section II describes unitdosage forms of the pharmaceutical compositions of the invention andmethods for administering them. Section III describes methods fortreating diseases and conditions by administration of the 2S,4Rketoconazole enantiomer and pharmaceutical compositions comprising the2S,4R ketoconazole enantiomer substantially free of the 2R,4Senantiomer.

I. Preparation of the 2S,4R Ketoconazole Enantiomer and PharmaceuticalCompositions Containing the 2S,4R Ketoconazole Enantiomer Substantiallyor Entirely Free of the 2R,4S Ketoconazole Enantiomer

As used herein, a composition containing “the 2S,4R ketoconazoleenantiomer substantially or entirely free of the 2R,4S ketoconazoleenantiomer” includes compositions that do not contain the 2R,4Sketoconazole enantiomer as well as compositions that containsubstantially less of the 2R,4S ketoconazole enantiomer, relative to theamount of the 2S,4R enantiomer, than do racemic ketoconazolecompositions currently approved for therapeutic use. Compositions usefulin the methods of the invention include, for example and withoutlimitation, compositions in which the total ketoconazole content iscomprised of at least 80%, or at least 90%, or at least 99%, or at least99.5%, or at least 99.9% or greater of the 2S,4R enantiomer.

The 2S,4R enantiomer of ketoconazole may be obtained by opticalresolution of racemic ketoconazole. Such resolution can be accomplishedby any of a number of resolution methods well known to a person skilledin the art, including but not limited to those described in Jacques etal., “Enantiomers, Racemates and Resolutions,” Wiley, New York (1981),incorporated herein by reference. For example, the resolution may becarried out by preparative chromatography on a chiral column. Anotherexample of a suitable resolution method is the formation ofdiastereomeric salts with a chiral acid such as tartaric, malic,mandelic acid or N-acetyl derivatives of amino acids, such as N-acetylleucine, followed by recrystallization to isolate the diastereomericsalt of the desired enantiomer. Yet another method for obtainingcompositions of the 2S,4R enantiomer substantially free of the 2R,4Senantiomer is a fractional crystallization of the diastereomeric salt ofketoconazole with (+)-camphor-10-sulfonic acid.

The 2S,4R enantiomer of ketoconazole can also be prepared directly by avariety of methods known to those of skill in the art. For example, the2S,4R enantiomer can be prepared directly by transketolization reactionsbetween 2-bromo-2′,4′-dichloroacetophenone and optically pure solketaltosylates, as described by Rotstein et al. (Rotstein et al., supra,incorporated herein by reference).

The present invention also provides a variety of pharmaceuticallyacceptable salts of the 2S,4R enantiomer of ketoconazole for use in thepharmaceutical compositions of the invention. The term “pharmaceuticallyacceptable salt” refers to salts prepared from pharmaceuticallyacceptable bases or acids, including inorganic or organic bases andinorganic or organic acids. Salts derived from inorganic bases includealuminum, ammonium, calcium, copper, ferric, ferrous, lithium,magnesium, manganic, manganous, potassium, sodium, and zinc salts, andthe like. The ammonium, calcium, magnesium, potassium, and sodium salts,in particular, can be preferred for some pharmaceutical formulations.Salts in the solid form can exist in more than one crystal structure andcan also be in the form of hydrates and polyhydrates. The solvates, and,in particular, the hydrates of the 2S,4R ketoconazole enantiomer areuseful in the preparation of the pharmaceutical compositions of thepresent invention.

Salts derived from pharmaceutically acceptable organic bases includesalts of primary, secondary and tertiary amines, substituted amines,including naturally occurring substituted amines, cyclic amines, andbasic ion exchange resins, such as arginine, betaine, caffeine, choline,N,N′-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol,2-dimethylamino ethanol, ethanolamine, ethylenediamine,N-ethyl-morpholine, N-ethylpiperidine, glutamine, glucosamine,histidine, hydrabamine, isopropylamine, lysine, methylglucamine,morpholine, piperazine, piperidine, polyamine resins, procaine, purines,theobromine, triethylamine, trimethylamine, tripropylamine, andtromethamine, and the like.

When the compound to be formulated is basic, salts can be prepared frompharmaceutically acceptable acids, including inorganic and organicacids. Such acids include acetic, benzenesulfonic, benzoic,camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic,hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic,methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric,succinic, sulfuric, tartaric, and p-toluenesulfonic acid, and the like.Illustrative pharmaceutically acceptable acids include citric,hydrobromic, hydrochloric, maleic, phosphoric, sulfuric, and tartaricacids. Ketoconazole compounds are often basic, because the triazole ringis basic. The 2S,4R ketoconazole compound can be made and handled as anon-pharmaceutically acceptable salt (e.g. trifluoroacetate salts)during synthesis and then converted as described herein to apharmaceutically acceptable salt.

Suitable pharmaceutically acceptable salts of the 2S,4R ketoconazoleenantiomer include, but are not limited to, the mesylate, maleate,fumarate, tartrate, hydrochloride, hydrobromide, esylate,p-toluenesulfonate, benzoate, acetate, phosphate, and sulfate salts. Forthe preparation of pharmaceutically acceptable acid addition salts ofthe compound of 2S,4R ketoconazole, the free base can be reacted withthe desired acids in the presence of a suitable solvent by conventionalmethods. Similarly, an acid addition salt can be converted to the freebase form by methods known to those of skill in the art.

Pharmaceutical compositions of the invention can include metabolites ofthe 2S,4R ketoconazole enantiomer that are therapeutically active orprodrugs of the enantiomer. Prodrugs are compounds that are converted totherapeutically active compounds as they are being administered to apatient or after they have been administered to a patient.

Thus, the pharmaceutical compositions of the invention comprise the2S,4R ketoconazole enantiomer, or a pharmaceutically acceptable salt,hydrate or solvate thereof, or a prodrug or active metabolite thereof,in combination with a pharmaceutically acceptable carrier andsubstantially or entirely free of the 2R,4S enantiomer. In oneembodiment, the pharmaceutical composition contains a therapeuticallyeffective amount of the 2S,4R enantiomer of ketoconazole or apharmaceutically acceptable salt thereof and a pharmaceuticallyacceptable carrier. As noted above, pharmaceutically acceptable salts ofthe 2S,4R enantiomer useful in such compositions include, but are notlimited to, the hydrochloride, phosphate, maleate, fumarate, tartrate,mesylate, esylate, and sulfate salts.

The “therapeutically effective amount” of the 2S,4R enantiomer ofketoconazole or pharmaceutically acceptable salt thereof will depend onthe condition to be treated, the route and duration of administration,the physical attributes of the patient, including weight and othermedications taken concurrently, and may be determined according tomethods well known to those skilled in the art in light of the presentdisclosure (see Section II, below). The pharmaceutical compositions ofthe invention can be conveniently prepared in unit dosage form bymethods well-known in the art of pharmacy as medicaments to beadministered orally, parenterally (including subcutaneous,intramuscular, and intravenous administration), ocularly (ophthalmicadministration), rectally, pulmonarily (nasal or oral inhalation),topically, transdermally or via buccal transfer.

The pharmaceutical compositions of the invention can be prepared bycombining the 2S,4R ketoconazole enantiomer with a selectedpharmaceutical carrier according to conventional pharmaceuticalcompounding techniques. Carriers take a wide variety of forms. Forexample, carriers for oral liquid compositions include, e.g., water,glycols, oils, alcohols, flavoring agents, preservatives, coloringagents and other components used in the manufacture of oral liquidsuspensions, elixirs and solutions. Carriers such as starches, sugarsand microcrystalline cellulose, diluents, granulating agents,lubricants, binders, disintegrating agents and the like are used toprepare oral solid dosage forms, e.g., powders, hard and soft capsulesand tablets. Solid oral preparations are typically preferred over oralliquid preparations.

Thus, in one embodiment, the pharmaceutically acceptable carrier is asolid and the pharmaceutical composition is a tablet for oraladministration. Other suitable forms of the pharmaceutical compositionsof the invention for oral administration include compressed or coatedpills, dragees, sachets, hard or soft gelatin capsules, sublingualtablets, syrups and suspensions. The oral solid dosage forms may alsocontain a binder such as gum tragacanth, acacia, corn starch, orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, or alginic acid; a lubricant such asmagnesium stearate; and/or a sweetening agent such as sucrose, lactose,or saccharin. Capsules may also contain a liquid carrier such as a fattyoil. Various other materials may be present to act as coatings or tomodify the physical form of the dosage unit. For instance, tablets maybe coated with shellac, sugar or both. Tablets may be coated by standardaqueous or nonaqueous techniques. The typical percentage of activecompound in these compositions may, of course, be varied from, forexample and without limitation, about 2 percent to about 60 percent on aw/w basis.

In another embodiment, the pharmaceutically acceptable carrier is aliquid, and the pharmaceutical composition is intended for oraladministration. Oral liquids suitable for use in such compositionsinclude syrups and elixirs and can contain, in addition to the activeingredient, sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye, and/or a flavoring, such as cherry or orangeflavor.

In another embodiment, the present invention provides a pharmaceuticalcomposition of the 2S,4R ketoconazole enantiomer suitable for parenteraladministration. For parenteral administration, the pharmaceuticalcomposition is typically contained in ampoules or vials and consistsessentially of an aqueous or non-aqueous solution or emulsion. Thesecompositions are typically in the form of a solution or suspension, andare typically prepared with water, and optionally include a surfactantsuch as hydroxypropylcellulose. Dispersions can be prepared in glycerol,liquid polyethylene glycols, and mixtures thereof in oils. Typically,preparations that are in diluted form also contain a preservative.

In another embodiment, the pharmaceutically acceptable carrier is aliquid, and the pharmaceutical composition is an injectable solution.The pharmaceutical injectable dosage forms, including aqueous solutionsand dispersions and powders for the extemporaneous preparation ofinjectable solutions or dispersions, are also sterile and, at the timeof administration, are sufficiently fluid for easy syringability. Thesecompositions are stable under the conditions of manufacture and storageand are typically preserved. The carrier thus includes the solvent ordispersion medium containing, for example, water, ethanol, polyol (e.g.glycerol, propylene glycol and liquid polyethylene glycol), suitablemixtures thereof, and vegetable oils.

In another embodiment, the pharmaceutically acceptable carrier is a gel,and the pharmaceutical composition is provided in the form of asuppository. For rectal administration, the pharmaceutical compositionis provided in a suppository, and the pharmaceutical acceptable carrieris a hydrophilic or hydrophobic vehicle. In another embodiment, thepharmaceutical composition useful in the methods of the invention isprepared for topical application, and the 2S,4R ketoconazole enantiomeris formulated as an ointment. The 2S,4R enantiomer can also beadministered transdermally; suitable transdermal delivery systems areknown in the art.

The pharmaceutical compositions of the invention also include sustainedrelease compositions. Suitable sustained release compositions includethose described in U.S. patent application publication Nos. 20050013834;20030190357; and 2002055512 and PCT patent application publication Nos.WO 03011258 and 0152833, each of which is incorporated herein byreference.

II. Unit Dosage Forms; Frequency and Duration of Administration

As noted above, any suitable route of administration can be employed forproviding a mammal, typically a human, but mammals of veterinaryimportance, such as cattle, horses, pigs, sheep, dogs, and cats, canalso benefit from the methods described herein, with a therapeuticallyeffective dose of the 2S,4R enantiomer. For example, oral, rectal,topical, parenteral, ocular, pulmonary, or nasal administration can beemployed. Dosage forms include tablets, troches, dispersions,suspensions, solutions, capsules, creams, ointments, aerosols and thelike. In many embodiments of the treatment methods of the invention, thepharmaceutical composition is administered orally. The therapeuticallyeffective dosage of the active ingredient varies depending on theparticular compound employed (salt, solvate, prodrug, or metabolite),the mode of administration, the condition being treated, and theseverity of the condition. Such dosages may be ascertained readily by aperson skilled in the art in light of the disclosure herein.

When treating or preventing the diseases and conditions as describedherein, satisfactory results can obtained when the 2S,4R ketoconazoleenantiomer is administered at a daily dosage of from about 0.1 to about25 milligrams (mg) per kilogram (mpk) of body weight, preferably givenas a single daily dose or in divided doses about two to six times a day.For oral administration to a human adult patient, the therapeuticallyeffective amount will generally be administered in the range of 50 mg to800 mg per dose, including but not limited to 100 mg per dose, 200 mgper dose, and 400 mg per dose, and multiple, usually consecutive dailydoses will be administered in a course of treatment. The 2S,4Rketoconazole enantiomer pharmaceutical composition can be administeredat different times of the day. In one embodiment the optimal therapeuticdose can be administered in the evening. In another embodiment theoptimal therapeutic dose can be administered in the morning. The totaldaily dosage of the 2S,4R ketoconazole enantiomer thus can in oneembodiment range from about 10 mg to about 2 g, and often ranges fromabout 10 mg to about 1 g, and most often ranges from about 100 mg toabout 500 mg. In the case of a typical 70 kg adult human, the totaldaily dose of the 2S,4R ketoconazole enantiomer can range from about 10mg to about 1000 mgs and will often range, as noted above, from about 50mg to about 800 mg. This dosage may be adjusted to provide the optimaltherapeutic response.

In one embodiment, the unit dosage form is suitable for oraladministration and contains one or more pharmaceutical excipients.Examples of pharmacologically inactive excipients that can be includedin an orally available formulation of the 2S,4R enantiomer ofketoconazole for purposes of the present invention and their functionare provided in the following table.

Inactive Ingredient Trade Name Grade Function Silicified Prosolv HD 90NF Diluent Microcrystalline Cellulose Lactose Modified, 316 Fast NFDiluent Monohydrate Flo Corn Starch STA-Rx NF Disintegrant MagnesiumStearate N/A NF Lubricant Colloidal Silicon Cab-O-Sil M5P NF GlidantDioxide

The excipients listed in the preceeding table can be combined in varyingproportion with the 2S,4R enantiomer to obtain specific drug tablet andmanufacturing characteristics. The drug tablet size can vary from 1 mgtotal weight to 1000 mg total weight; for example and withoutlimitation, from 100 mg total weight to 800 mg total weight. Theproportion of the 2S,4R enantiomer present in the drug tablet can varyfrom 1% to 100%; for example and without limitation, from 10% to 90%. Anexample of a 400 mg tablet with the 2S,4R enantiomer comprising 50% ofthe tablet weight is provided in the following table. In this example,dry blends were made with the (−) cis 2S,4R ketoconazole and the listedinactive excipients and pressed as a dry blend into tablets.

Tablet Weight Component % w/w (mg) (−)cis 2S,4R 50.0 200 KetoconazoleLactose Monohydrate, 22.4 89.6 NF Silicified 16.5 66.0 MicrocrystallineCellulose, NF Corn Starch, NF 10.0 40.0 Colloidal Silicon 0.5 2.0Dioxide, NF) Magnesium Stearate, NF 0.6 2.4 Total 100.0 400.0

A drug tablet formulation for 2S,4R ketoconazole was described in U.S.Pat. No. 6,040,307. This formulation included the active drug substance,(−) ketoconazole, Lactose, Cornstarch, water and Magnesium Stearate. Wetgranules were generated with the ketoconazole, lactose, water and cornstarch, these granules were dried in an oven prior to compressing intotablets with magnesium stearate and more corn starch. Tablets werecompressed and dried. This is a less optimal method than the method ofthe invention described above using a dry blend process, as excess waterand elevated temperatures are not introduced. Ketoconazole can undergodegradation (oxidation) (Farhadi and Maleki (2001). “A newspectrophotometric method for the determination of ketoconazole based onthe oxidation reactions.” Analytical Sciences 17 Supplement, i867-i869.The Japan Society for Analytical Chemistry), and oxidation reactions areaccelerated in the presence of water and elevated temperatures.

The solid unit dosage forms of the pharmaceutical compositions of theinvention contain the 2S,4R ketoconazole enantiomer or a salt or hydratethereof in an amount ranging from about 1 mg to about 2 g, often fromabout 1.0 mg to about 1.0 g, and more often from about 10 mg to about500 mg. In the liquid pharmaceutical compositions of the inventionsuitable for oral administration, the amount of the 2S,4R ketoconazoleenantiomer can range from about 1 mg/ml to about 200 mg/ml. Thetherapeutically effective amount can also be an amount ranging fromabout 10 mg/ml to about 100 mg/ml. In one embodiment, the dose of theliquid pharmaceutical composition administered is an amount between 0.5ml and 5.0 ml. In another embodiment, the dose is between about 1 ml and3 ml. In the liquid pharmaceutical compositions of the inventiondesigned for intravenous or subcutaneous administration the amount ofthe 2S,4R ketoconazole the amount of the 2S,4R enantiomer can range fromabout 0.01 to 1 mg/ml and can be administered at a rate of between 0.01to 1 ml/minute by either a subcutaneous or intravenous administration.Alternatively the amount of the 2S,4R enantiomer can range from about0.1 mg/ml to 10 mg/ml and can be administered at a rate of between 0.001ml/minute to 0.1 ml/minute by either of a subcutaneous or intravenousadministration.

As noted above, the pharmaceutical compositions of the invention willtypically be administered for multiple consecutive days for periodsranging from one or more weeks to one, several, or many months (e.g., atleast 7, 14, 28, 60 or 120 days). In one embodiment, the pharmaceuticalcompositions of the invention are administered for the treatment of achronic disease, condition, or indication for treatment periods rangingfrom one month to twelve months. In another embodiment, the 2S,4Renantiomer is administered from one year to five years. In anotherembodiment, the 2S,4R enantiomer is administered from 5 years to 20years. In another embodiment, the 2S,4R enantiomer is administered untilthere is remission from the disease or for the life of the patient.

The duration of administration in accordance with the methods of theinvention depends on the disease or condition to be treated, the extentto which administration of the pharmaceutical composition hasameliorated the disease symptoms and conditions, and the individualpatient's reaction to the treatment.

III. Methods for Treating Diseases and Conditions with thePharmaceutical Compositions of the Invention

Inhibition of Cortisol Synthesis

The 2S,4R enantiomer of ketoconazole is significantly more effective perweight unit at lowering the plasma concentration of physiologicallyactive glucocorticoids than is either the racemic ketoconazole or theother enantiomer in racemic ketoconazole, the 2R,4S enantiomer. Inaddition, and as demonstrated in the Figures and in the Examples below,and as distinct from racemic ketoconazole, the 2S,4R enantiomer does notcause a time dependent increase in exposure to the 2S,4R enantiomer.Thus, the methods of the present invention offer significant therapeuticbenefit over methods involving the administration of racemicketoconazole in the treatment of diseases and conditions associated withelevated levels or aberrant activity of cortisol or in the treatment ofdiseases in which a benefit can be obtained by lowering normal cortisollevels or activity.

Cortisol promotes both the accumulation of adipose tissue and therelease of free fatty acids from adipose tissue. When oxidized, freefatty acids act in an antagonistic manner to insulin in the liver,reducing insulin sensitivity in the liver (i.e., increasing hepaticinsulin resistance). Cortisol also acts directly as an antagonist to theaction of insulin in the liver, such that insulin sensitivity is furtherreduced. Cortisol also directly increases the amount of the ratelimiting enzymes controlling glucose production by the liver. Theseactions result in increased gluconeogenesis and elevated levels ofglucose production by the liver. Hepatic insulin resistance also resultsin impaired lipoprotein synthesis by the liver and so is a majorcontributing factor to the dyslipidemia known in patients with type 2diabetes and in patients with Metabolic Syndrome. Patients who alreadyhave impaired glucose tolerance have a greater probability of developingtype 2 diabetes in the presence of abnormally high levels of cortisol.High levels of cortisol can also lead to hypertension, in part throughactivation of the mineralocorticoid receptor. Inhibition of 11β-HSD-Ienzyme shifts the ratio of cortisol and cortisone in specific tissues infavour of cortisone. The 2S,4R ketoconazole enantiomer is a cortisolsynthesis inhibitor acting on the 11β hydroxylase enzyme and may alsoexert its therapeutic effect, at least in part, by inhibition of the11β-HSD-I enzyme.

The present invention provides methods for using the 2S,4R enantiomer ofketoconazole, a cortisol synthesis inhibitor, for the treatment,control, amelioration, prevention, delay in the onset of or reduction ofthe risk of developing the diseases and conditions due at least in partto cortisol and/or other corticosteroids in a mammalian patient,particularly a human. In one embodiment, the method involves theadministration of a therapeutically effective amount of the 2S,4Rketoconazole enantiomer or a pharmaceutically acceptable salt or solvatethereof, substantially or entirely free of other ketoconazoleenantiomers, to the patient suffering from the disease or condition.

Cortisol activity can contribute to a large number of diseases andconditions, including, but not limited to, type 2 diabetes, metabolicsyndrome, obesity, dyslipidemia, insulin resistance, and hypertension.These and other diseases and conditions susceptible to treatment withthe compositions of the invention in accordance with the methods of theinvention are described below.

Diabetes, Metabolic Syndrome, and Related Diseases and Conditions

Diabetes is caused by multiple factors and is most simply characterizedby elevated levels of plasma glucose (hyperglycemia) in the fastingstate. There are two generally recognized forms of diabetes: type 1diabetes, in which patients produce little or no insulin, the hormonewhich regulates glucose production and utilization, and type 2 diabetes,in which patients produce insulin and even exhibit hyperinsulinemia(plasma insulin levels that may be similar or even elevated incomparison with non-diabetic subjects), while at the same timedemonstrating hyperglycemia. Patients with type 2 diabetes typicallyhave some degree of resistance to the glucose lowering actions ofinsulin. Type 1 diabetes is typically treated with exogenous insulinadministered via injection.

However, patients with type 2 diabetes typically develop “insulinresistance”, such that the effect of insulin in stimulating glucose andlipid metabolism in the main insulin-sensitive tissues, namely, muscle,liver, and adipose tissues, is diminished. Patients who are insulinresistant but do not have diabetes have elevated insulin levels thatcompensate for their insulin resistance, so that serum glucose levelsare not elevated. In patients with type 2 diabetes, the plasma insulinlevels, even when they are elevated, are insufficient to overcome thepronounced insulin resistance, resulting in hyperglycemia. Patients withtype 2 diabetes may also have elevated circulating cortisol levelsand/or production rates (see Lee et al., “Plasma insulin, growthhormone, cortisol, and central obesity among young Chinese type 2diabetic patients.” Diabetes Care 1999; 22(9): 1450-7; Homma et al.,“Assessing systemic 11β-hydroxysteroid dehydrogenase with serumcortisone/cortisol ratios in healthy subjects and patients with diabetesmellitus and chronic renal failure.” Metabolism 2001; 50(7): 801-4; andRichardson and Tayek, “Type 2 diabetic patients may have a mild form ofan injury response: a clinical research center study.” Am J PhysiolEndocrinol Metab 2002; 282(6): E1286-90; Chiodini et al. “Association ofsubclinical hypercortisolism with type 2 diabetes mellitus: acase-control study in hospitalized patients.” Eur J Endocrinol 2005;153(6): 837-844; Liu et al. “Elevated late-night salivary cortisollevels in elderly male type 2 diabetic veterans.” Clin Endocrinol (Oxf)2005; 63(6): 642-9; and Catargi et al. “Occult Cushing's syndrome intype-2 diabetes.” J Clin Endocrinol Metab 2003; 88(12): 5808-13, each ofwhich is incorporated herein by reference). Excess cortisol is now known(see U.S. Pat. No. 5,849,740, incorporated herein by reference) toinduce insulin resistance and two prime characteristics of type 2diabetes: reduced peripheral glucose uptake and increased hepaticglucose output. See also Rizza et al., “Cortisol-induced insulinresistance in man: impaired suppression of glucose production andstimulation of glucose utilization due to a postreceptor defect ofinsulin action.” J Clin Endocrinol Metab 1982; 54(1): 131-8; Holmang andBjorntorp, “The effects of cortisol on insulin sensitivity in muscle.”Acta Physiol Scand 1992; 144(4): 425-31; Lecavalier et al.,“Glucagon-cortisol interactions on glucose turnover and lactategluconeogenesis in normal humans.” Am J Physiol 1990; 258(4 Pt 1):E569-75; and Khani and -Tayek, “Cortisol increases gluconeogenesis inhumans: its role in the metabolic syndrome.” Clin Sci (Lond) 2001;101(6): 739-47; each of which is incorporated herein by reference.

Persistent or uncontrolled hyperglycemia that occurs in diabetes isassociated with increased morbidity and premature mortality. Abnormalglucose homeostasis is also associated both directly and indirectly withobesity, hypertension, and alterations in lipid, lipoprotein, andapolipoprotein metabolism. Patients with type 2 diabetes are atincreased risk of cardiovascular complications, e.g., atherosclerosis,coronary heart disease, stroke, peripheral vascular disease,hypertension, nephropathy, neuropathy and retinopathy. Therefore,therapeutic control of glucose homeostasis, lipid metabolism, obesity,and hypertension are critically important in the clinical management andtreatment of diabetes mellitus. The present invention provides methodsfor such therapeutic control by the administration of therapeuticallyeffective amounts of the 2S,4R enantiomer of ketoconazole substantiallyor entirely free of the 2R,4S enantiomer.

Many patients who have insulin resistance but have not (yet) developedtype 2 diabetes are also at a risk of developing a constellation ofsigns or symptoms previously referred to as the “Insulin ResistanceSyndrome, Dysmetabolic Syndrome or Syndrome X”, now more widely known asthe “Metabolic Syndrome”. Metabolic Syndrome is characterized by insulinresistance, along with abdominal obesity, hyperinsulinemia, high bloodpressure, low HDL levels, high VLDL triglyceride and small dense LDLparticles and elevated glucose levels. These patients, whether or notthey develop overt diabetes mellitus, are at increased risk ofdeveloping the cardiovascular complications listed above. Patients withMetabolic Syndrome have been reported to have abnormalities in cortisollevels, production or catabolism (see Berceanu-Gabrielescu et al.,“Hypercorticism—a risk factor in arterial hypertension andatherosclerosis.” Endocrinologie 1981; 19(2): 123-7; Phillips et al.,“Elevated plasma cortisol concentrations: a link between low birthweight and the insulin resistance syndrome?” J Clin Endocrinol Metab1998; 83(3): 757-60; and Ward et al., “Cortisol and the metabolicsyndrome in South Asians.” Clin Endocrinol (Oxf) 2003; 58(4): 500-5;each of which is incorporated herein by reference).

Treatment of type 2 diabetes typically includes diet therapy andincreased physical exercise either alone or in combination withpharmacologic therapy. Increasing the plasma level of insulin byadministration of sulfonylureas (e.g. tolbutamide, and glipizide) ormeglitinides, which Stimulate the pancreatic beta cells to secrete moreinsulin, and/or by injection of insulin when sulfonylureas ormeglitinides become ineffective, can result in insulin concentrationshigh enough to stimulate insulin-resistant tissues. However, dangerouslylow levels of plasma glucose can result, and an increased level ofinsulin resistance can ultimately occur.

Biguanides reduce excessive production of glucose by the liver andincrease insulin sensitivity, resulting in some correction ofhyperglycemia. However, many biguanides, e.g., phenformin and metformin,can cause lactic acidosis, nausea, and diarrhea.

The thiazolidinediones or glitazones (i.e.5-benzylthiazolidine-2,4-diones) are a newer class of compounds thathave been characterized as having potential for amelioratinghyperglycemia and other symptoms of type 2 diabetes. These agentsincrease insulin sensitivity in muscle, liver, and adipose tissue,resulting in partial or complete correction of the elevated plasmalevels of glucose substantially without causing hypoglycemia. Theglitazones that are currently marketed are agonists of the peroxisomeproliferator activated receptor (PPAR) γ subtype. PPARγ agonism isgenerally believed to be responsible for the improved insulinsensitization that is observed with the glitazones. Newer PPAR agoniststhat are being developed for treatment of type 2 diabetes and/ordyslipidemia are agonists of one or more of the PPAR α, γ and δsubtypes. One disadvantage of all known glitazones is theirweight-increasing effect, mediated via an increase in adipose tissuemass. Another disadvantage is that glitazones have been associated withan increased risk of heart failure, mediated via fluid retention.

There remains a need for new methods of treating diabetes and relatedconditions, such as the various conditions that individually andcollectively contribute to Metabolic Syndrome. The present inventionmeets this need. The present invention provides a method of treatingdiabetes, and the related conditions of hyperglycemia and insulinresistance in a mammalian patient in need of such treatment, whichmethod comprises administering to said patient a therapeuticallyeffective amount of a pharmaceutical composition containing the 2S,4Renantiomer of ketoconazole substantially free of the 2R,4S enantiomer.In one embodiment, the method is used to treat type 2 diabetes.Administration of a therapeutically effective amount of an11β-hydroxylase inhibitor such as the 2S,4R ketoconazole enantiomersubstantially free of the 2R,4S enantiomer is effective in treating,controlling, and ameliorating the symptoms of diabetes, particularlytype 2 diabetes, and administration of a therapeutically effectiveamount of an 11β-hydroxylase inhibitor such as the 2S,4R ketoconazoleenantiomer substantially free of the 2R,4S enantiomer on a regular,daily basis can delay or prevent the onset of type 2 diabetes.

By reducing insulin resistance and maintaining serum glucose at normalconcentrations, the pharmaceutical compositions of this invention alsohave utility in the treatment and prevention of conditions thataccompany type 2 diabetes and insulin resistance, including obesity(typically abdominal obesity), Metabolic Syndrome (“Syndrome X”),including each of the symptoms and conditions that contribute to thesyndrome, diabetic retinopathy, neuropathy, nephropathy, and prematurecardiovascular disease.

Excessive levels of cortisol have been associated with obesity, whichmay be associated with the ability of cortisol to stimulate adipogenesisin general and visceral (also known as abdominal) obesity in particular.Visceral/abdominal obesity is closely associated with glucoseintolerance, hyperinsulinemia, hypertriglyceridemia, and other factors(conditions and symptoms) of Metabolic Syndrome, such as high bloodpressure, elevated VLDL and reduced HDL, as well as diabetes. Thus, theadministration of an effective amount of an 11β-hydroxylase inhibitorsuch as the 2S,4R ketoconazole enantiomer substantially free of the2R,4S enantiomer is useful in the treatment or control of obesity (e.g.,abdominal obesity) and Metabolic Syndrome. Long-term treatment with an11β-hydroxylase inhibitor such as the 2S,4R ketoconazole enantiomersubstantially free of the 2R,4S enantiomer in accordance with themethods of the invention is also useful in delaying or preventing theonset of obesity, especially if the patient uses an 11β-hydroxylaseinhibitor such as the 2S,4R ketoconazole enantiomer substantially freeof the 2R,4S enantiomer in combination with controlled diet andexercise.

Thus, in another embodiment, the present invention provides a method oftreating obesity (e.g., abdominal obesity) in a mammalian patient inneed of such treatment, which method comprises administering to saidpatient a therapeutically effective amount of the 2S,4R ketoconazoleenantiomer substantially free of the 2R,4S enantiomer. Likewise, inanother embodiment, the present invention provides a method of treatingMetabolic Syndrome in a mammalian patient in need of such treatment,which comprises administering to said patient a therapeuticallyeffective amount of a pharmaceutical composition containing the 2S,4Rketoconazole enantiomer substantially free of the 2R,4S enantiomer.

Atherosclerosis, Lipid Disorders, Hypertension

Inhibition of 14α lanosterol demethylase and a reduction in cholesteroland inhibition of 11β-hydroxylase activity and a reduction in the amountof cortisol are beneficial in treating or controlling hypertension anddyslipidemia. Because hypertension and dyslipidemia contribute to thedevelopment of atherosclerosis, administration of a therapeuticallyeffective amount of a 14α-lanosterol demethylase inhibitor and an11β-hydroxylase inhibitor such as the 2S,4R ketoconazole enantiomersubstantially free of the 2R,4S enantiomer can be beneficial intreating, controlling, delaying the onset of, or preventinghypertension, dyslipidemia, and atherosclerosis. In one embodiment, theinvention provides a method of treating atherosclerosis in a mammalianpatient in need of such treatment, said method comprising administeringto said patient a therapeutically effective amount of a pharmaceuticalcomposition containing the 2S,4R ketoconazole enantiomer substantiallyfree of the 2R,4S enantiomer.

In another embodiment, the present invention provides a method oftreating a lipid disorder selected from the group consisting ofdyslipidemia, hyperlipidemia, hypertriglyceridemia,hypercholesterolemia, low HDL, and high LDL, in a mammalian patient inneed of such treatment, such method comprising administering to saidpatient a therapeutically effective amount of a pharmaceuticalcomposition containing the 2S,4R ketoconazole enantiomer substantiallyfree of the 2R,4S enantiomer.

Stroke

Inhibition of 14α lanosterol demethylase and a reduction in cholesteroland inhibition of 11β-hydroxylase activity and a reduction in the amountof cortisol are beneficial in treating or ischemic stroke. Becausecortisol, hypertension and dyslipidemia contribute to the severity andmortality of ischemic strokes, administration of a therapeuticallyeffective amount of a 14α-lanosterol demethylase inhibitor and an11β-hydroxylase inhibitor such as the 2S,4R ketoconazole enantiomersubstantially free of the 2R,4S enantiomer can be beneficial intreating, or reducing the severity of ischemic strokes. In oneembodiment, the invention provides a method of treating an ischemicstroke event in a patient in need of such treatment, said methodcomprising administering to said patient a therapeutically effectiveamount of a pharmaceutical composition containing the 2S,4R ketoconazoleenantiomer substantially free of the 2R,4S enantiomer

Alzheimer's Disease

Inhibition of 14α lanosterol demethylase and a reduction in cholesteroland inhibition of 11β-hydroxylase activity and a reduction in the amountof cortisol are beneficial in treating or Alzheimer's disease. Becauseelevated cortisol has been associated with the development ofAlzheimer's disease and a reduction in cholesterol through the use ofstatins may reduce the severity of Alzheimer's disease, administrationof a therapeutically effective amount of a 14α-lanosterol demethylaseinhibitor and an 11β-hydroxylase inhibitor such as the 2S,4Rketoconazole enantiomer substantially free of the 2R,4S enantiomer canbe beneficial in treating, or reducing the severity of Alzheimer'sdisease. In one embodiment, the invention provides a method of treatingAlzheimer's disease in a mammalian patient in need of such treatment,said method comprising administering to said patient a therapeuticallyeffective amount of a pharmaceutical composition containing the 2S,4Rketoconazole enantiomer substantially free of the 2R,4S enantiomer.

Cognitive Impairment, Dementia, and Depression

Excessive levels of cortisol in the brain can also result in neuronalloss or dysfunction through the potentiation of neurotoxins. Cognitiveimpairment has been associated with aging and excess levels of cortisolin the brain (see Seckl Walker, “Minireview: 11β-hydroxysteroiddehydrogenase type 1—a tissue-specific amplifier of glucocorticoidaction.” Endocrinology 2001; 142(4): 1371-6, incorporated herein byreference). Administration of an effective amount of an 11β-hydroxylaseinhibitor such as the 2S,4R ketoconazole enantiomer substantially freeof the 2R,4S enantiomer results in the reduction, amelioration, control,or prevention of cognitive impairment associated with aging and ofneuronal dysfunction. In one embodiment, the invention provides a methodof treating cognitive impairment, neuronal dysfunction, and/or dementiain a mammalian patient in need of such treatment, said method comprisingadministering to said patient a therapeutically effective amount of apharmaceutical composition of the 2S,4R ketoconazole enantiomersubstantially free of the 2R,4S enantiomer.

Another condition in which high cortisol levels are reported to becausally important is depression. Muck-Seler et al. (Muck-Seler et al.,“Platelet serotonin and plasma prolactin and cortisol in healthy,depressed and schizophrenic women.” Psychiatry Res 2004; 127(3): 217-26,incorporated herein by reference) reported that plasma cortisol levelswere significantly increased both in schizophrenic and in depressedpatients compared with values in healthy controls. In one embodiment,the invention provides a method of treating depression in a mammalianpatient in need of such treatment, said method comprising administeringto said patient a therapeutically effective amount of a pharmaceuticalcomposition of the 2S,4R ketoconazole enantiomer substantially free ofthe 2R,4S enantiomer.

Cushing's Syndrome

Cushing's Syndrome is a metabolic disease or condition in which patientshave high cortisol levels in their blood stream. These high levels mayresult from adrenal gland malfunction due to a pituitary tumor or asecondary tumor, both producing the cortisol secretagogue ACTH in excessor be due to a tumor or disorder of the adrenal gland per se thatdirectly overproduces cortisol. Patients with Cushing's syndrome oftendevelop type 2 diabetes. Treatment of Cushing's Syndrome can involveremoval of the offending tumor and/or treatment with cortisol synthesisinhibitors such as metyrapone, ketoconazole, or aminoglutethimide (seeMurphy, “Steroids and depression.” J Steroid Biochem Mol Biol 1991;38(5): 537-59, incorporated herein by reference). In one embodiment, thepresent invention provides a method of treating Cushing's Syndrome in apatient in need of such treatment, which method comprises administeringto said patient a therapeutically effective amount of a pharmaceuticalcomposition of the 2S,4R ketoconazole enantiomer substantially free ofthe 2R,4S enantiomer, alone or in combination with another cortisolsynthesis inhibitor, such as metyrapone or aminoglutethimide.

Decreased Insulin Secretion

Glucocorticoids have been shown to reduce insulin secretion in vivo (seeBillaudel and Sutter, “Direct effect of corticosterone upon insulinsecretion studied by three different techniques.” Horm Metab Res 1979;11(10): 555-60, incorporated herein by reference). Inhibition ofcortisol synthesis as provided by the pharmaceutical compositions usedin the methods of the invention can therefore be beneficial in thetreatment of decreased insulin secretion. In addition, reduced11beta-HSD-I activity has been observed, in isolated murine pancreaticbeta cells, to improve glucose stimulated insulin secretion (see Davaniet al., “Type 1 11beta-hydroxysteroid dehydrogenase mediatesglucocorticoid activation and insulin release in pancreatic islets.” JBiol Chem 2000; 275(45): 34841-4, incorporated herein by reference). Inone embodiment, the invention provides a method of treating decreasedinsulin secretion in a mammalian patient in need of such treatment, saidmethod comprising administering to said patient a therapeuticallyeffective amount of a pharmaceutical composition of the 2S,4Rketoconazole enantiomer substantially free of the 2R,4S enantiomer.

Glaucoma and Intraocular Pressure

There is a connection between the levels of glucocorticoid targetreceptors and the 11□-HSD-I enzymes and the susceptibility to glaucoma(see Stokes et al., “Altered peripheral sensitivity to glucocorticoidsin primary open-angle glaucoma.” Invest Ophthalmol Vis Sci 2003; 44(12):5163-7, incorporated herein by reference). High cortisol levels arereported to be causally important in glaucoma. Median total plasma,plasma free, and percent free cortisol levels are higher in patient withocular hypertension and glaucoma. The most significant differencesoccurred with percent free cortisol values between normal andglaucomatous subjects (see Schwartz et al., “Increased plasma freecortisol in ocular hypertension and open angle glaucoma.” ArchOphthalmol 1987; 105(8): 1060-5, incorporated herein by reference).

In accordance with the methods of the present invention, inhibition of11β-hydroxylase activity by the administration of the 2S,4R ketoconazoleenantiomer substantially free of the 2R,4S enantiomer is useful inreducing intraocular pressure and in the treatment of glaucoma. In oneembodiment, the invention provides a method of treating glaucoma andreducing intraocular pressure in a mammalian patient in need of suchtreatment, said method comprising administering to said patient atherapeutically effective amount of a pharmaceutical composition of the2S,4R ketoconazole enantiomer substantially free of the 2R,4Senantiomer.

Immunomodulation

In certain disease states, such as tuberculosis, psoriasis, and evenunder conditions of excessive stress, high glucocorticoid activityshifts the immune response to a humoral response, when in fact a cellbased response may be more beneficial to the patient. Inhibition of11β-HSD-I activity and the attendant reduction in glucocorticoid levelsshifts the immune response toward a cell based response (see Mason,“Genetic variation in the stress response: susceptibility toexperimental allergic encephalomyelitis and implications for humaninflammatory disease.” Immunol Today 1991; 12(2): 57-60; and Rook,“Glucocorticoids and immune function.” Baillieres Best Pract Res ClinEndocrinol Metab 1999; 13(4): 567-81; each of which is incorporatedherein by reference). In one embodiment, the invention provides a methodof modulating the immune response to a cell-based response in amammalian patient in need of such treatment, said method comprisingadministering to said patient a therapeutically effective amount of apharmaceutical composition of the 2S,4R ketoconazole enantiomersubstantially free of the 2R,4S enantiomer.

Impaired Renal Function.

Increased intra-renal blood pressure can lead to renal damage. Cortisolcan compete with true mineralocorticoids for access to the aldosteronereceptor and increase blood pressure. Ketoconazole has been tested inpatients with renal failure and has been shown to increase glomerularfiltration rate. Ketoconazole has also been shown to decrease theleakage of albumin from kidneys in patients with diabetes type 2 withoutrenal failure. Thus, in one embodiment the invention provides a methodof treating impaired renal function or reducing albumin leakage in amammalian patient in need of such treatment, said method comprisingadministering to said patient a therapeutically effective amount of apharmaceutical composition of the 2S,4R ketoconazole enantiomersubstantially free of the 2R,4S enantiomer

Therapeutic Uses of the 2S,4R Ketoconazole Eenantiomer

In view of the foregoing, those of skill in the art will appreciate thatthe present invention provides a method of treating a condition selectedfrom the group consisting of: (1) hyperglycemia, (2) low glucosetolerance, (3) insulin resistance, (4) obesity, (5) lipid disorders, (6)dyslipidemia, (7) hyperlipidemia, (8) hypertriglyceridemia, (9)hypercholesterolemia, (10) low HDL levels, (11) high LDL levels, (12)atherosclerosis and its sequelae, (13) vascular restenosis, (14)pancreatitis, (15) abdominal obesity, (16) neurodegenerative disease,(17) retinopathy, (18) nephropathy, (19) neuropathy, (20) MetabolicSyndrome, and (21) other conditions and disorders where insulinresistance is a component, in a mammalian patient in need of suchtreatment, said method comprising administering to the patient atherapeutically effective amount of a pharmaceutical composition of the2S,4R ketoconazole enantiomer substantially free of the 2R,4Senantiomer.

In another aspect, the present invention provides a method of delayingthe onset of a condition selected from the group consisting of (1)hyperglycemia, (2) low glucose tolerance, (3) insulin resistance, (4)obesity, (5) lipid disorders, (6) dyslipidemia, (7) hyperlipidemia, (8)hypertriglyceridemia, (9) hypercholesterolemia, (10) low HDL levels,(11) high LDL levels, (12) atherosclerosis and its sequelae, (13)vascular restenosis, (14) pancreatitis, (15) abdominal obesity, (16)neurodegenerative disease, (17) retinopathy, (18) nephropathy, (19)neuropathy, (20) Metabolic Syndrome, and (21) other conditions anddisorders where insulin resistance is a component in a mammalian patientin need of such treatment, said method comprising administering to thepatient a therapeutically effective amount of a pharmaceuticalcomposition of the 2S,4R ketoconazole enantiomer substantially free ofthe 2R,4S enantiomer.

In another aspect, the present invention provides a method of reducingthe risk of developing a condition selected from the group consisting of(1) hyperglycemia, (2) low glucose tolerance, (3) insulin resistance,(4) obesity, (5) lipid disorders, (6) dyslipidemia, (7) hyperlipidemia,(8) hypertriglyceridemia, (9) hypercholesterolemia, (10) low HDL levels,(11) high LDL levels, (12) atherosclerosis and its sequelae, (13)vascular restenosis, (14) pancreatitis, (15) abdominal obesity, (16)neurodegenerative disease, (17) retinopathy, (18) nephropathy, (19)neuropathy, (20) Metabolic Syndrome, and (21) other conditions anddisorders where insulin resistance is a component in a mammalian patientin need of such treatment, said method comprising administering to thepatient a therapeutically effective amount of a pharmaceuticalcomposition of the 2S,4R ketoconazole enantiomer substantially free ofthe 2R,4S enantiomer.

Other Conditions

The invention provides a method for reducing plasma cortisol levels in asubject not diagnosed with or under treatment for a fungal infection, byadministering a pharmaceutical composition comprising a pharmaceuticallyacceptable carrier and a therapeutically effective amount of 2S,4Rketoconazole enantiomer substantially free of the 2R,4S ketoconazoleenantiomer to the subject. For example, the methods of the invention mayalso be used for treatment of diseases and conditions in which cortisollevels are not elevated (e.g., normal or below normal levels) but inwhom therapeutic benefit can be obtained by reducing cortisol levels.

Additional Optional Subject Characteristics

In certain aspects of the invention, a patient being treated with apharmaceutical composition comprising the 2S,4R ketoconazole enantiomersubstantially free of the 2R,4S enantiomer is not diagnosed with and/oris not under treatment for a fungal infection. In certain aspects of theinvention, a patient being treated with a pharmaceutical compositioncomprising the 2S,4R ketoconazole enantiomer substantially free of the2R,4S enantiomer is not diagnosed with and/or is not under treatment forhypercholesterolemia. In certain aspects of the invention, a patientbeing treated with a pharmaceutical composition of the 2S,4Rketoconazole enantiomer substantially free of the 2R,4S enantiomer isnot diagnosed with and/or is not under treatment for one or morediseases, disorders, or conditions independently selected from thefollowing: (1) hyperglycemia, (2) low glucose tolerance, (3) insulinresistance, (4) obesity, (5) a lipid disorder, (6) dyslipidemia, (7)hyperlipidemia, (8) hypertriglyceridemia, (9) hypercholesterolemia, (10)low HDL levels, (11) high LDL levels, (12) atherosclerosis (12)atherosclerosis and its sequelae, (13) vascular restenosis, (14)pancreatitis, (15) abdominal obesity, (16) neurodegenerative disease,(17) retinopathy, (18) nephropathy, (19) neuropathy, (20) MetabolicSyndrome, (21) prostate cancer, (22) benign prostatic hyperplasia, and(23) other conditions and disorders where insulin resistance is acomponent.

Reducing Cortisol Levels in a Subject by Providing a Constant Exposureto1-acetyl-4-[4-[[2-(2,4-dichlorophenyl)-2-[(1H-imidazol-1-yl)-methyl]-1,3-dioxolan-4-yl]methoxy]phenyl]piperazine

In one aspect the invention provides a method of reducing cortisollevels in a subject by providing a constant exposure to1-acetyl-4-[4-[[2-(2,4-dichlorophenyl)-2-[(1H-imidazol-1-yl)-methyl]-1,3-dioxolan-4-yl]methoxy]phenyl]piperazineby administering doses of 2S,4R enantiomer that are substantially freeof the 2R,4S enantiomer to the patient. In this context, providing aconstant exposure to1-acetyl-4-[4-[[2-(2,4-dichlorophenyl)-2-[(1H-imidazol-1-yl)-methyl]-1,3-dioxolan-4-yl]methoxy]phenyl]piperazinemeans that the drug does not accumulate in the subject to whom the drugis administered.

In one embodiment the 2S,4R is administered over a period of at least 14days (e.g., 14 days), and preferably at least 28 days (e.g., 28 days).In one embodiment, the doses of 2S,4R enantiomer are administered daily(as a single or multiple daily administration). In one embodiment, thedoses of 2S,4R enantiomer are administered on alternate days. In oneembodiment, the doses of 2S,4R enantiomer are administered according toan other schedule as part of a course of therapy, where the course oftherapy lasts at least 28 days and where administration of an equalweight amount (or, alternatively, a double weight amount) of racemicketoconazole results in accumulation of the drug in the subject.

Accumulation of drug, or the absence of accumulation, can be measured bydetermining the plasma level of drug on a first day and on a measuringthe plasma level of the drug on one or more subsequent days. Forexample, if the plasma level is measured on a first day, denoted Day 1,subsequent measurements can be made on Day 7 and/or Day 14 and/or Day28, or daily for 1, 2 or 4 weeks. In one embodiment, determining theplasma level involves measuring a 12 hour or 24 hour AUC. In oneembodiment, the cortisol plasma level on Day 1 and on at least onesubsequent day selected from Day 7, Day 14 and Day 28 differs by lessthan about 50%, preferably by less than about 25%, and sometimes by lessthan 15%. It will be appreciated that, guided by this disclosure, aconstant exposure of a particular subject to1-acetyl-4-[4-[[2-(2,4-dichlorophenyl)-2-[(1H-imidazol-1-yl)-methyl]-1,3-dioxolan-4-yl]methoxy]phenyl]piperazinecan also be deduced from administration of doses shown inpharmacokinetic studies to result in constant exposure in astatistically significant number of similar subjects.

In a preferred embodiment, the constant exposure is provided byadministering a constant total periodic dose of the 2S4R enantiomer,such as a constant total daily dose (in one or more administrations perday). In an embodiment, the subject has not previously been treated withracemic or enantiomeric ketoconazole. In one embodiment, the subject hasnot been administered drug for at least 14 days, at least 28 days, or atleast 6 months prior to Day 1. In one embodiment the subject is a humanpatient. In another embodiment, the subject is a dog or is aSprague-Dawley rat. In an embodiment, the subject is diagnosed with acondition characterized by elevated cortisol levels.

Combination Therapies

Thus, a variety of diseases, disorders, and conditions can be treated,controlled, prevented or delayed with the pharmaceutical compositionsand methods of this invention, including but not limited to: (1)hyperglycemia, (2) low glucose tolerance, (3) insulin resistance, (4)obesity, (5) lipid disorders, (6) dyslipidemia, (7) hyperlipidemia, (8)hypertriglyceridemia, (9) hypercholesterolemia, (10) low HDL levels,(11) high LDL levels, (12) atherosclerosis and its sequelae, (13)vascular restenosis, (14) pancreatitis, (15) abdominal obesity, (16)neurodegenerative disease, (17) retinopathy, (18) nephropathy, (19)neuropathy, (20) Metabolic Syndrome, and (21) other disorders whereinsulin resistance is a component. In one embodiment, a method of theinvention is practiced on a patient who concurrently receives anothertreatment for one or more of these conditions.

As is apparent from the Figures and the Examples provided herein the2S,4R enantiomer of ketoconazole does not alter the pharmacokinetics ofthe 2S,4R enantiomer and, by extension, the 2S,4R enantiomer ofketoconazole will not alter the pharmacokinetics of other drugs that aremetabolized and excreted by the same pathways that are utilized by the2S,4R enantiomer. Thus, the present invention provides for a method ofco-administering drugs that are commonly co-administered with racemicketoconazole without the risks of aberrant pharmacokinetics of theco-administered drug or racemic ketoconazole attendant to theadministration of racemic ketoconazole.

The pharmaceutical compositions of the invention can be co-administeredor otherwise used in combination with one or more other drugs in thetreatment, prevention, suppression, or amelioration of the diseases,disorders, and conditions described herein as susceptible to therapeuticintervention in accordance with the methods of the invention. Typically,the combination of the drugs provided by the methods of the presentinvention is safer or more effective than either drug alone or of thenon-2S,4R ketoconazole enantiomer drug in combination with racemicketoconazole, or the combination is safer or more effective than wouldbe expected based on the additive properties of the individual drugs.Such other drugs) may be administered by a route and in an amountcommonly used contemporaneously or sequentially with a pharmaceuticalcomposition of the 2S,4R ketoconazole enantiomer substantially free ofthe 2R,4S enantiomer. When a pharmaceutical composition of the 2S,4Rketoconazole enantiomer substantially free of the 2R,4S enantiomer isused contemporaneously with one or more other drugs, a combinationproduct containing such other drug(s) and the 2S,4R ketoconazoleenantiomer can be utilized if the two active drugs can be coformulated.Combination therapy in accordance with the methods of the invention alsoincludes therapies in which the pharmaceutical compositions useful inthe methods of the invention and one or more other drugs areadministered on different overlapping schedules. It is contemplatedthat, when used in combination with other active ingredients, thepharmaceutical compositions useful in the methods of the presentinvention or the other active ingredient or both may be used effectivelyin lower doses than when each is used alone. Accordingly, thepharmaceutical compositions useful in the methods of the presentinvention include those that contain one or more other activeingredients, in addition to the 2S,4R ketoconazole enantiomer.

Examples of other drugs that may be administered in combination with apharmaceutical composition of the present invention, either separatelyor, in some instances, the same pharmaceutical composition, include, butare not limited to:

(a) dipeptidyl peptidase IV (DPP-IV) inhibitors; (b) insulin sensitizersincluding (i) PPARγ agonists such as the glitazones (e.g. pioglitazone,rosiglitazone, and the like) and other PPAR ligands, including PPARα/γdual agonists, such as KRP-297, and PPARαagonists such as gemfibrozil,clofibrate, fenofibrate and bezafibrate, and (ii) biguanides, such asmetformin and phenformin; (c) insulin, insulin analogs, or insulinmimetics;

(d) sulfonylureas and other insulin secretagogues such as tolbutamide,glipizide, glyburide, meglitinide, and related materials; (e)α-glucosidase inhibitors (such as acarbose); (f) glucagon receptorantagonists such as those disclosed in PCT patent applicationpublication Nos. WO 98/04528, WO 99/01423, WO 00/39088, and WO 00/69810,each of which is incorporated herein by reference; (g) GLP-1, GLP-1analogs and mimetics, and GLP-1 receptor agonists such as thosedisclosed in PCT patent application publication Nos. WO 00/42026 and WO00/59887, each of which is incorporated herein by reference; (h) GIP,GIP analogs and mimetics, including but not limited to those disclosedin PCT patent application publication No. WO 00/58360, incorporatedherein by reference, and GIP receptor agonists; (i) PACAP, PACAP analogsand mimetics, and PACAP receptor 3 agonists such as those disclosed inPCT patent application publication No. WO 01/23420, incorporated hereinby reference; (j) cholesterol lowering agents such as (i) HMG-CoAreductase inhibitors (lovastatin, simvastatin, pravastatin, fluvastatin,atorvastatin, rivastatin, itavastatin, rosuvastatin, and other statins),(ii) sequestrants (cholestyramine, colestipol, and dialkylaminoalkylderivatives of a cross-linked dextran), (iii) nicotinyl alcohol,nicotinic acid or a salt thereof, (iv) inhibitors of cholesterolabsorption, such as for example ezetimibe and β-sitosterol, (v) acylCoA:cholesterol acyltransferase inhibitors, such as for exampleavasimibe, and (vi) anti-oxidants such as probucol; (k) PPARδ agonists,such as those disclosed in PCT patent application publication No. WO97/28149, incorporated herein by reference; (1) antiobesity compoundssuch as fenfluramine, dexfenfluramine, phentermine, sibutramine,orlistat, neuropeptide Y5 inhibitors, CB1 receptor inverse agonists andantagonists, and β₃ adrenergic receptor agonists; (m) an ileal bile acidtransporter inhibitor; (n) agents intended for use in inflammatoryconditions other than glucocorticoids, such as aspirin, non-steroidalanti-inflammatory drugs, azulfidine, and cyclooxygenase 2 selectiveinhibitors, and (o) protein tyrosine phosphatase-1B (PTP-1B) inhibitors.

Thus, in one embodiment, the present invention provides a pharmaceuticalcomposition that comprises: (1) a therapeutically effective amount of2S,4R ketoconazole enantiomer substantially free of 2R,4S ketoconazoleenantiomer; (2) a therapeutically effective amount of compound selectedfrom the group consisting of: (a) DPP-IV inhibitors; (b) insulinsensitizers selected from the group consisting of (i) PPAR agonists and(ii) biguanides; (c) insulin and insulin analogs and mimetics; (d)sulfonylureas and other insulin secretagogues; (e) α-glucosidaseinhibitors; (f) glucagon receptor antagonists; (g) GLP-1, GLP-1 analogsand mimetics, and GLP-1 receptor agonists; (h) GP, GIP analogs andmimetics, and GIP receptor agonists; (i) PACAP, PACAP analogs andmimetics, and PACAP receptor 3 agonists; (j) cholesterol lowering agentsselected from the group consisting of (i) HMG-CoA reductase inhibitors,(ii) sequestrants, (iii) nicotinyl alcohol, nicotinic acid or a saltthereof, (iv) PPARα agonists, (v) PPARα/γ dual agonists, (vi) inhibitorsof cholesterol absorption, (vii) acyl CoA:cholesterol acyltransferaseinhibitors, and (viii) anti-oxidants; (k) PPARδ agonists; (1)antiobesity compounds; (m) an ileal bile acid transporter inhibitor; (n)anti-inflammatory agents other than glucocorticoids; and (o) proteintyrosine phosphatase-1B (PTP-1B) inhibitors; and (3) a pharmaceuticallyacceptable carrier.

The above pharmaceutical compositions and combination therapies includethose in which the 2S,4R ketoconazole enantiomer substantially orentirely free of the 2R,4S enantiomer, or a pharmaceutically acceptablesalt, hydrate, or solvate thereof, is co-formulated or co-administeredwith one or more other active compounds. Non-limiting examples includecombinations of the 2S,4R ketoconazole enantiomer with two or moreactive compounds selected from biguanides, sulfonylureas, HMG-CoAreductase inhibitors, PPAR agonists, PTP-1B inhibitors, DPP-IVinhibitors, and anti-obesity compounds.

Thus, in one embodiment, the present invention provides a method oftreating a condition selected from the group consisting of (1)hyperglycemia, (2) low glucose tolerance, (3) insulin resistance, (4)obesity, (5) lipid disorders, (6) dyslipidemia, (7) hyperlipidemia, (8)hypertriglyceridemia, (9) hypercholesterolemia, (10) low HDL levels,(11) high LDL levels, (12) atherosclerosis and its sequelae, (13)vascular restenosis, (14) pancreatitis, (15) abdominal obesity, (16)neurodegenerative disease, (17) retinopathy, (18) nephropathy, (19)neuropathy, (20) Metabolic Syndrome, and (21) other conditions anddisorders where insulin resistance is a component, in a mammalianpatient in need of such treatment, said method comprising administeringto the patient therapeutically effective amounts of a pharmaceuticalcomposition of the 2S,4R ketoconazole enantiomer substantially free ofthe 2R,4S enantiomer and of a compound or pharmaceutical compositioncomprising said compound selected from the group consisting of: (a)DPP-IV inhibitors; (b) insulin sensitizers selected from the groupconsisting of (i) PPAR agonists and (ii) biguanides; (c) insulin andinsulin analogs mimetics; (d) sulfonylureas and other insulinsecretagogues; (e) α-glucosidase inhibitors; (f) glucagon receptorantagonists; (g) GLP-1, GLP-1 analogs and mimetics, and GLP-1 receptoragonists; (h) GIP, GIP analogs and mimetics, and GIP receptor agonists;(i) PACAP, PACAP analogs and mimetics, and PACAP receptor 3 agonists;(j) cholesterol lowering agents selected from the group consisting of(i) HMG-CoA reductase inhibitors, (ii) sequestrants, (iii) nicotinylalcohol, nicotinic acid and salts thereof, (iv) PPARα agonists, (v)PPARα/γ dual agonists, (vi) inhibitors of cholesterol absorption, (vii)acyl CoA:cholesterol acyltransferase inhibitors, and (viii)anti-oxidants; (k) PPARδ agonists; (1) antiobesity compounds; (m) anileal bile acid transporter inhibitor; (n) anti-inflammatory agentsexcluding glucocorticoids; and (o) protein tyrosine phosphatase-1B(PTP-1B) inhibitors.

In another embodiment, the present invention provides a method oftreating a condition selected from the group consisting ofhypercholesterolemia, atherosclerosis, low HDL levels, high LDL levels,hyperlipidemia, hypertriglyceridemia, and dyslipidemia, in a mammalianpatient in need of such treatment, said method comprising administeringto the patient a therapeutically effective amount of a pharmaceuticalcomposition of the 2S,4R ketoconazole enantiomer substantially free ofthe 2R,4S enantiomer and an HMG-CoA reductase inhibitor. In oneembodiment, the HMG-CoA reductase inhibitor is a statin. In oneembodiment, the statin is selected from the group consisting oflovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin,itavastatin, ZD-4522, rosuvastatin, and rivastatin.

In another embodiment, the present invention provides a method ofreducing the risk of developing a condition selected from the groupconsisting of hypercholesterolemia, atherosclerosis, low HDL levels,high LDL levels, hyperlipidemia, hypertriglyceridemia and dyslipidemia,and the sequelae of such conditions is disclosed comprisingadministering to a mammalian patient in need of such treatment atherapeutically effective amount of a pharmaceutical composition of the2S,4R ketoconazole enantiomer substantially free of the 2R,4S enantiomerand an HMG-CoA reductase inhibitor. In another embodiment, the methodfor delaying the onset or reducing the risk of developingatherosclerosis in a human patient in need of such treatment furthercomprises the administration of a cholesterol absorption inhibitor incombination with a statin HMG-CoA reductase inhibitor and apharmaceutical composition of the 2S,4R ketoconazole enantiomersubstantially free of the 2R,4S enantiomer. In one embodiment, thecholesterol absorption inhibitor is a cholesterol transfer ester protein(CTEP) inhibitor. In another embodiment the CTEP inhibitor is ezetimibe.

In another embodiment, the invention provides a method for delaying theonset or reducing the risk of developing atherosclerosis in a humanpatient in need of such treatment, said method comprising administeringto said patient an effective amount of a pharmaceutical composition ofthe 2S,4R ketoconazole enantiomer substantially free of the 2R,4Senantiomer and an HMG-CoA reductase inhibitor. In one embodiment, theHMG-CoA reductase inhibitor is a statin. In one embodiment, the statinis selected from the group consisting of: lovastatin, simvastatin,pravastatin, fluvastatin, atorvastatin, itavastatin, ZD-4522,rosuvastatin and rivastatin. In one embodiment, the statin issimvastatin.

The invention, numerous embodiments of which have been described above,may be further appreciated and understood by the examples below, whichdemonstrate that the 2S,4R enantiomer is more effective than racemicketoconazole or the 2R,4S enantiomer in the racemate at reducing theconcentration of the active glucocorticoid in the plasma and does notimpair (or impairs to a significantly less extent) drug metabolism asdoes racemic ketoconazole.

EXAMPLES Example 1 Measurement of Corticosterone and CholesterolFollowing Dosing with Racemic Ketoconazole and the Enantiomers ofKetoconazole

The effect of ketoconazole and the ketoconazole enantiomers oncorticosterone levels in the plasma of Sprague Dawley rats wasdetermined. For the experiment described in FIG. 1, the four enantiomersand the racemic ketoconazole were suspended in olive oil. To generatethe results shown in FIG. 1, five groups (eight per group) of rats wereused. The rats were maintained on a 14/10 hour light/dark cycle andallowed free access to food and water. Each rat was dosed (200 mgdrug/kg body weight) via a gastric tube. The rats in group 1 were dosedwith the vehicle (olive oil), while the rats in the other four groupswere dosed with one of the four ketoconazole enantiomers as indicated.All of the rats were dosed between 2.00 and 3.00 pm and were sacrificedfour hours later (between 6.00 and 7.00 pm). Plasma was prepared and theconcentration of corticosterone determined by an enzyme linked immunoassay (ELISA). In rats, the predominant active glucocorticoid iscorticosterone; in humans, the predominant active glucucorticoid is theclosely related molecule cortisol. The results shown in FIG. 1demonstrate that, in comparison to the vehicle control, the two transenantiomers (2S45 and 2R4R), when given to rats at 200 mg/kg, havelittle effect on the blood level of corticosterone. In contrast, bothcis enantiomers reduce corticosterone, with the 2S,4R beingsignificantly more efficacious than 2R,4S.

For the experiment summarized in FIG. 2, there was one vehicle (oliveoil) group of 9 rats, and 15 groups of 10 rats/group treated with thespecified dose of ketoconazole or one of the two (2S,4R and 2R,4S) cisenantiomers of ketoconazole. The rats were maintained and dosed asdescribed above. Plasma was prepared and the concentration ofcorticosterone in the plasma determined by ELISA. The results shown inFIG. 2 demonstrate that there is a dose dependent effect of bothketoconazole and the enantiomers on corticosterone levels and that the2S,4R enantiomer is significantly more efficacious than both theketoconazole racemate and the other cis enantiomer (2R,4S).

For the experiment summarized in FIG. 3 and FIG. 8, there were sixgroups of ten rats/group that were treated with the vehicle (olive oil)and eighteen groups of 10 rats/group treated with either ketoconazole orone of the two (2S,4R and 2R,4S) cis enantiomers of ketoconazole. Therats were maintained as described above; the drugs were suspended inolive oil, and each rat was dosed once via gastric tube to achieve adose of 200 mg/kg. All of the rats were dosed at a specific time so thatall terminations occurred between 6 and 7 pm. For example, the ratstreated for 24 hours were dosed between 6 and 7 pm the day prior tosacrifice, and the rats treated for 12 hours were dosed between 6 and 7am on the day of sacrifice. Following sacrifice, plasma was prepared,and the concentration of corticosterone in the plasma was determined byELISA. In the same plasma samples, total cholesterol levels were alsodetermined. The results shown in FIG. 3 demonstrate that the 2S,4R issignificantly more efficacious than the 2R,4S enantiomer at loweringcorticosterone and that this increased efficacy persists for at least 24hours. The efficacy of the racemate is intermediate between the twoenantiomers. Similarly, the results shown in the table below demonstratethat the 2S,4R is significantly more efficacious than the 2R,4Senantiomer at lowering cholesterol. The results show that efficacy ofthe racemate is intermediate between the two enantiomers.

Effect of racemic ketoconazole and the 2S,4R, and 2R,4S enantiomers oncholesterol levels in rats at the indicated time after oral dosing with200 mg of the indicated drug (or vehicle) Cholesterol Levels (mean ±SEM; mg/dL) Time (hours) Vehicle 2S,4R (DIO-902) 2R,4S Racemate  4 77.3± 3.9 69.6 ± 1.9 85.1 ± 7 81.2 ± 3.9  8 73.5 ± 0.5 73.5 ± 3.1 85.1 ± 5.473.5 ± 2.3 12 69.6 ± 3.5 77.3 ± 3.9 77.3 ± 1.9 69.6 ± 2.3 16 69.6 ± 1.961.9 ± 3.1 77.3 ± 4.6 69.6 ± 3.1 20 69.6 ± 1.9   58 ± 1.2 69.6 ± 2.765.7 ± 2.7 24 65.7 ± 2.7 61.9 ± 3.1 69.6 ± 1.5 65.7 ± 3.9

Example 2 Measurement of Drug Exposure Following Dosing with RacemicKetoconazole and the Cis Enantiomers of Ketoconazole

In this example, dogs were treated with ketoconazole or with the 2S,4Renantiomer only, and the plasma levels of the corresponding drug weredetermined.

Pharmacokinetics of Racemic Ketoconazole

Two groups of three male and three female dogs per group were studied.Each dog was dosed with racemic ketoconazole, and the concentration ofracemic ketoconazole in the plasma of the dogs was determined on thefirst day of dosing and again after four weeks of daily dosing. The twogroups differed in that, in one group, the racemic ketoconazole wasprovided as a dry white powder in a gelatin capsule, and in the other,the racemic ketoconazole was provided as a suspension in olive oil.

The dogs were purpose bred beagle dogs obtained from Covance ResearchProducts, Inc., Cumberland, Va. USA. The dogs were 4.5 to 5 months oldat the start of dosing. The dogs were housed in suspended, stainlesssteel cages. Air conditioning provided a minimum of 10 air changes/hour.The temperature and relative humidity ranges were 18 to 29 degreeCentigrade and 30% to 70%, respectively. With a few exceptions whenmanual over-ride was used for study related activities, fluorescentlighting was controlled automatically to give a cycle of 12 hours light(0700-1900) and 12 hours dark. Certified canine diet (#8727C, HarlanTeklad) was available ad libitum. Water was provided ad libitum via anautomatic watering system. After arrival at the test lab, the dogs wereacclimated for 19 days and then randomized, as needed, to a treatmentgroup using a computerized blocking procedure designed to achieve bodyweight balance. After allocation, the mean body weights were calculatedand inspected to ensure there were no unacceptable differences betweengroups. The animals were individually identified by means of anelectronic implant.

In the first group, the dogs were dosed daily by oral delivery of agelatin capsule (size 13, Torpac, N.J., USA). The capsule containedsufficient racemic ketoconazole to provide a dose of 40 mg drug/kg bodyweight/day. The capsules were prepared weekly for each animal based onindividual body weights. The capsules and the bulk drug were stored atroom temperature in sealed containers. For the second group, the gelatincapsules contained sufficient racemic ketoconazole suspended in oliveoil to provide a dose of 40 mg drug/kg body weight/day. The animals wereobserved approximately 1 to 2 hours after dosing, daily, throughout theexperiment. Blood samples (1 ml into lithium heparin) were taken fromthe jugular vein from each of the animals on the first day of dosing andagain at week 4 (after 28 daily doses) at 0 (pre-dose) 1, 2, 4, 8, 12,and 24 hours after dosing. At week 4, the pre-dose sample was timed tobe 24 hours post-dosing on the previous day. Plasma samples were storedfrozen at −70 degrees Centigrade until analysis. The plasma samples wereanalyzed for racemic ketoconazole as described below using racemicketoconazole as a standard.

As shown in FIG. 4, the pharmacokinetic profile (concentration as afunction of time) of racemic ketoconazole in the plasma of the dogsdosed only once (and the plasma assayed over the first 24 hours afterdosing) was significantly diminished as compared to the pharmacokineticprofile of racemic ketoconazole in the plasma of dogs dosed daily for 28days (and the plasma assayed over the 24 hours after the last of the 28doses). This effect was obtained in both groups (racemic ketoconazoleadministered as a dry powder and racemic ketoconazole administered as asuspension in olive oil). The Area Under the Curve (AUC) was calculatedusing the linear trapezoidal rule. The AUC determined after a singledose was significantly reduced in comparison to the AUC determined after28 daily doses (see FIG. 5). Again, this effect was seen in both groups(racemic ketoconazole administered as a dry powder and racemicketoconazole administered as a suspension in olive oil).

Pharmacokinetics of the 2S,4R Enantiomer

Another group of three female and three male dogs was dosed with the2S,4R enantiomer of ketoconazole, and the concentration of theenantiomer in the plasma of the dogs was determined on the first day ofdosing and again after four weeks of daily dosing.

The dogs were purpose bred beagle dogs obtained from Harlan, Bicester,Kent, England. The dogs were 4.5 to 5 months old and weighed between 6.7and 8.85 kg on arrival at the test lab. They were approximately 6 to 6.5months of age at the start of dosing. The dogs were housed in a singleexclusive room, air conditioned to provide a minimum of 10 airchanges/hour. The temperature and relative humidity ranges were 16 to 24degree Centigrade and 30% to 80%, respectively. With a few exceptionswhen manual over-ride was used for study related activities, fluorescentlighting was controlled automatically to give a cycle of 12 hours light(0700-1900) and 12 hours dark. The animals were housed singly during theday in pens of 2.25 m², and animals of the same experimental group andsex were housed overnight in pens of at least 4.5 m².

Each animal was offered 400 g of Harlan Teklad Dog Maintenance Diet(Harlan, Teklad, Bicester, England) and a Winalot Shapes biscuit(Friskies Pet Care, Suffolk, England) each morning after dosing withketoconazole or the 2 S4R enantiomer. Water was provided ad libitum viaan automatic watering system. Bedding was provided on a daily basis toeach animal by use of clean wood flakes/shavings (Datesand Ltd.Manchester, England). After arrival at the test lab, the dogs wereacclimated for 7 weeks and then randomized, as needed, to a treatmentgroup based on a stratified randomization procedure, using littermatedata and the most recent body weight data. After allocation, the meanbody weights were calculated and inspected to ensure there were nounacceptable differences between groups. The animals were individuallyidentified by means of an electronic implant.

Three male and three female dogs were dosed daily by oral delivery of agelatin capsule (size 13, Torpac, N.J., USA). The capsule containedsufficient 2S,4R enantiomer to provide a dose of 20 mg drug/kg bodyweight/day. The capsules were prepared weekly for each animal based onindividual body weights. The capsules and the bulk drug were stored atroom temperature in sealed containers. The animals were observedapproximately 1 to 2 hours after dosing, daily throughout theexperiment. Blood samples (1 ml into lithium heparin) were taken fromthe jugular vein from each of the animals on the first day of dosing andagain at week 4 (after 28 daily doses) at 0 (pre-dose) 1, 2, 4, 8, and24 hours after dosing. At week 4, the pre-dose sample was timed to be 24hours post-dosing on the previous day. Plasma samples were stored frozenat −70 degrees Centigrade until analysis. The plasma samples wereanalyzed for the 2S,4R enantiomer as described below using racemicketoconazole as a standard.

As shown in FIG. 6, the pharmacokinetic profile (concentration as afunction of time) of the 2S,4R enantiomer in the plasma of the dogsdosed only once (and the plasma assayed over the first 24 hours afterdosing) was not distinguishable from the pharmacokinetic profile of the2S,4R enantiomer in the plasma of dogs dosed daily for 28 days (and theplasma assayed over the 24 hours after the last of the 28 doses). TheArea Under the Curve (AUC) was calculated using the linear trapezoidalrule. The AUC determined after a single dose was not distinguishablefrom the AUC determined after 28 daily doses (see FIG. 7).

Ketoconazole Assay Procedures

Assays were established and validated using racemic ketoconazole. Plasmafrom the dogs treated with racemic ketoconazole, the 2S,4R enantiomer,or the vehicle control was prepared by standard methods and frozen at−70 degrees Centigrade until assayed. To assay the concentration ofracemic ketoconazole (or the 2S,4R enantiomer), the plasma samples werethawed and briefly vortexed, and 100 microliter aliquots taken. Aninternal standard (clotrimazole 25 microliters, 100 micrograms/mL, SigmaAldrich) was added to the samples and mixed briefly. The samples weresubjected to solid phase extraction using OASIS HLB (Waters Ltd. 730-740Centennial Court, Centennial Park, Elstree, Hertsfordshire WD6 3SZEngland). The eluates were evaporated to dryness under a stream ofnitrogen at nominal 40 degrees Centigrade and the residues re-dissolvedin a mobile phase prior to analysis by liquid chromatography withultraviolet light detection.

Concentrations of racemic ketoconazole and ketoconazole 2S,4R enantiomerin calibration standards and study samples were determined using leastsquares regression with reciprocal of the concentration (1/x) asweighting to improve accuracy at low levels. The lower limit ofquantification (LLOQ) for ketoconazole in dog plasma was 0.25micrograms/milliliter with linearity demonstrable to 25micrograms/milliliter. The coefficients of determination (r²) werebetter than or equal to 0.99226.

Example 3 Formulation and Clinical Trial of the 2S,4R EnantiomerSubstantially Free of the 2R,4S Enantiomer of Ketoconazole in Type 2Diabetes

A. Abbreviations

The following abbreviations are used in this Example.

Term/Abbreviation Explanation ALT alanine transaminase AST aspartatetransaminase AUC area under the curve Bid twice daily Biw twice weeklyBUN blood urea nitrogen CV coefficient of variation ELISA enzyme-linkedimmunosorbent assay FDA Food and Drug Administration GI GastrointestinalGLP Good Laboratory Practice IND Investigational New Drug (application)IV Intravenous MedDRA Medical Dictionary for Regulatory Activities NDANew Drug Application NOAEL no-observed-adverse-effect level PBSphosphate-buffered saline Qd Daily Qw Weekly RP-HPLC reverse-phasehigh-performance liquid chromatography SBA Summary Basis of Approval SCsubcutaneous, subcutaneously SD standard deviation SDS-PAGE sodiumdodecyl sulfate-polyacrylamide gel electrophoresis SE-HPLCsize-exclusion high-performance liquid chromatography USP United StatesPharmacopoeia WBC white blood cellB. Overview

An illustrative formulation of the 2S,4R enantiomer of ketoconazolesubstantially free of the 2R,4S enantiomer (hereinafter called DIO-902)is described in this Example together with pre-clinical data supportingits testing as an investigational new drug in human clinical trials forthe treatment of the hyperglycemia associated with type 2 diabetesmellitus. All references cited herein are incorporated herein byreference. Secondary benefits of this drug candidate are expected toinclude reduced total and LDL cholesterol, reduced blood pressure andreduced visceral adiposity. Racemic ketoconazole (the mixture of the twoenantiomers 2S,4R and 2R,4S) is an approved drug (NIZORAL®) for thetreatment of a variety of fungal infections. As racemic ketoconazolealso inhibits cortisol synthesis, this drug is used as a non-approvedtherapy for patients with Cushing's syndrome. In these patients racemicketoconazole reduces glucose, cholesterol, and blood pressure. Ascortisol may be a contributing causal factor in the development of type2 diabetes, clinical trials with racemic ketoconazole have been carriedout in these patients. The results of these clinical trials supporttreating type 2 diabetes through lowering of plasma cortisol. Racemicketoconazole has, however, been associated with hepatotoxicity.Preclinical results support that DIO-902 may be safer and moreefficacious than the racemic mixture.

DIO-902 is the 2S,4R enantiomer of ketoconazole (2S,4Rcis-1-acetyl-4-[4-[[2-(2,4-dichlorophenyl)-2-(1H-imidazol-1-ylmethyl)-1,3-dioxolan-4-yl]methoxyl]phenyl]piperazine).Ketoconazole, an approved drug, is a racemic mixture of both the 2S,4Renantiomer and the 2R,4S enantiomer. DIO-902 has been purified from theracemic mixture and is largely (greater than 99%) free of the 2R,4Senantiomer. It is anticipated that the primary pharmacological effect ofDIO-902 will be through the suppression of cortisol synthesis, withsecondary benefits exerted through a reduction in cholesterol synthesis.DIO-902 has been formulated into immediate release tablets. Thetoxicology of DIO-902 has been tested in dogs. At oral doses of up to 20mg/kg/day for 28 days the only noted effect was a reduction in foodintake and a reduction in body weight and a trend to a decrease incholesterol. There were no noted changes in any of the other serumchemistry or the hematological parameters measured. Higher single doseshave been used in rats. At 200 mg drug/kg body weight DIO-902 suppressestestosterone to 10% of basal. The suppression occurs within four hoursof dosing and testosterone levels return to normal within 8 hours.DIO-902 is orally available and reaches a maximal plasma concentrationbetween 2 and 8 hours in dogs. DIO-902 at 200 mg drug/kg body weightreduces serum levels of the active glucocorticoid in rodents(corticosterone) to 25% of basal within 4 hours of oral dosing. Thisdose of drug also suppresses plasma cholesterol. Thus, DIO-902 (2S,4R)is significantly more potent with respect to reducing corticosterone inrats than is the other enantiomer (2R,4S) and is more potent withrespect to reducing cholesterol in rats than is the other enantiomer.

DIO-902 has not been previously administered as a single chemical entityto human patients. However, this molecule has been widely administeredas part of the approved racemic mixture. When normal volunteers aregiven the racemic mixture, both enantiomers are orally available, and,after a 200 mg dose, a maximum plasma concentration of the DIO-902(approximately 3.6 μg/mL) is reached at 2 hours. The approved use forthe racemic mixture is for the treatment of fungal infections and theapproved dose is 200 mg BID. In addition, higher doses of the racemicmixture (up to 2000 mg/day) have been used. The racemic mixture has alsobeen used for non-approved indications, including Cushing's syndrome andprostate cancer. The racemic mixture can cause hepatoxicity and reducestestosterone, and 1,25 dihydroxy Vitamin D.

The diagnostic criterion for type 2 diabetes is hyperglycemia.Specifically, the American Diabetes Association recognizes a diagnosisof diabetes in which the patient displays one of the following threecharacteristics: a) a casual (any time of day or night) plasma glucosevalue of greater than 200 mg/dL (11.1 mmol/L) on two separate occasionsin presence or absence of the symptoms of diabetes (polyuria, polydipsiaor unexplained weight loss), or b) a fasting (8 hour) plasma glucosevalue of greater than 126 mg/dl (7 mmol/L), or c) a plasma glucose valueof greater than 200 mg/dl (11.1 mmol/L) 2 hours after a 75 gram oralload of glucose. Prospective studies have convincingly demonstrated thathyperglycemia is causally associated with long term microvascularcomplications including nephropathy and retinopathy. In addition to thediagnostic hyperglycemia, patients with type 2 diabetes have anincreased incidence of hypertension, hypertriglyceridemia andhypercholesterolemia. These significantly increase the risk ofmacrovascular and microvascular diseases.

The most important acquired risk factor for the development of type 2diabetes is adiposity, more specifically, visceral adiposity. There arealso genetic susceptibilities. Except for a small number of clearlydefined syndromes such as Maturity Onset Diabetes of the Young (MODY,principally caused by mutations in the gene encoding glucokinase) mostof the genes that contribute to the development of type 2 diabetes havenot been identified. Physiologically, hyperglycemia in patients withtype 2 diabetes is caused primarily by insulin resistance—a relativefailure of insulin to stimulate glucose uptake and to suppress glucoseproduction. This insulin resistance is initially partially compensatedfor by increased insulin synthesis. In many patients there is a laterstage where insulin production declines with a significant worsening ofthe hyperglycemia. There is still some uncertainty surrounding the causeof the insulin resistance with evidence supporting key roles forintracellular lipids and direct alterations in the activity of insulinsignaling molecules. Increased glucocorticoid bioactivity could also bea direct or indirect cause of insulin resistance and beta cell failure.

An important treatment option in patients with type 2 diabetes isdietary modification, increased exercise and weight loss. Unfortunately,while effective, this option has proved difficult to implement.Pharmacological therapeutics include metformin, sulphonylureas (andMeglitinide and Nateglinide which, like the sulphonylureas increaseinsulin secretion), the glitizides (Pioglitizone and Rosiglitizone) andinsulin. Although effective, glucose control remains sub-optimal. In2005, at their annual conference, The American Association of ClinicalEndocrinologists (AACE) announced a new glycosylated hemoglobin standardof 6.5% or lower for patients with type 2 diabetes. This new standard ispart of an effort to prevent diabetes complications. Dr. Paul Jellinger,current president of the American College of Endocrinology (ACE), saidthat the AACE is embarking on this effort after a study showed twothirds of Americans with type 2 diabetes are not adequately treating thedisease. Further, none of the drugs approved for glucose control appearto target the underlying cause(s) of the insulin resistance and some(such as insulin and the glitizones) can cause weight gain, which mayexacerbate the insulin resistance. One advantage of DIO-902 overcurrently available therapeutics is that this drug is believed totargets one of the causes of type 2 diabetes.

An additional potential advantage of DIO-902 is the possibility thatthis drug is believed to be able to improve significantly othercardiovascular risk factors including hypercholesterolemia andhypertension. The majority of patients with type 2 diabetes havecoexisting cardiovascular risk factors, including hypertension,dyslipidemia, and microalbuminuria (Alexander et al. (2003).“NCEP-defined metabolic syndrome, diabetes, and prevalence of coronaryheart disease among NHANES III participants age 50 years and older.”Diabetes 52(5): 1210-4). Independent of glycemic control, controllinghypertension and microalbuminuria has been shown to prevent both themicro- and macrovascular complications of diabetes. Further, the controlof dyslipidemia contributes to cardiovascular risk reduction and maydecrease the risk of developing diabetic nephropathy (Bell (2002).“Chronic complications of diabetes.” South Med J95(1): 30-4). Racemicketoconazole reduces blood pressure and cholesterol in patients withCushing's syndrome (Sonino et al. (1991). “Ketoconazole treatment inCushing's syndrome: experience in 34 patients.” Clin Endocrinol (Oxf)35(4): 347-52) and reduces cholesterol in patients withhypercholesterolemia (Gylling et al. (1993). “Effects of ketoconazole oncholesterol precursors and low density lipoprotein kinetics inhypercholesterolemia.” J Lipid Res 34(1): 59-67) and prostate cancer(Miettinen (1988). “Cholesterol metabolism during ketoconazole treatmentin man.” J Lipid Res 29(1): 43-51). Data obtained in the Phase 2clinical trial described by IND 60,874 also support that racemicketoconazole reduces total and LDL cholesterol and blood pressure inpatients with type 2 diabetes. Preclinical results described here and inExample 1 indicate that DIO-902 will have enhanced activities withrespect to blood pressure and cholesterol.

As noted above, the behavioral and therapeutic options available forpatients with type 2 diabetes are inadequate. The life style changeshave proved very difficult to implement. The therapeutic options do nottarget the underlying cause(s) of the disease and some therapies, forexample insulin and the glitizones, may exacerbate factors such as bodyweight that contribute to the underlying insulin resistance. Further,most therapeutic options reduce one (hyperglycemia), or at most two(hyperglycemia and either of hypertension or dyslipidemia) of thefactors that contribute to the micro and macro vascular complications.DIO-902 is believed to target an important causal component of type 2diabetes (elevated cortisol bioactivity) and to be able to treat thehyperglycemia, hypertension and dyslipidemia in these patients.

That glucocorticoids can decrease insulin sensitivity and increaseplasma glucose levels through effects on the liver, fat, muscle andpancreatic beta cells in humans (as well as in experimental animals) iswell established (McMahon et al. (1988). “Effects of glucocorticoids oncarbohydrate metabolism.” Diabetes Metab Rev 4(1): 17-30). In rodentmodels, glucocorticoids are necessary for the development of obesity,glucose intolerance and diabetes and, in some cases increasedglucocorticoid activity is sufficient to cause diabetes. In humans,pathological increases in glucocorticoid levels (as seen in patientswith Cushing's syndrome) can also cause diabetes. More recently there isa growing recognition that patients with incidental adrenal tumors(incidentalomas) and more subtle increases in cortisol activity are atsignificantly elevated risk for developing diabetes, glucose intolerancehypertension, diffuse obesity and dyslipidemia (Terzolo et al. (1998).“Subclinical Cushing's syndrome in adrenal incidentaloma.” ClinEndocrinol (Oxf) 48(1): 89-97; Rossi et al. (2000). “SubclinicalCushing's syndrome in patients with adrenal incidentaloma: clinical andbiochemical features.” Clin Endocrinol Metab 85(4): 1440-8).

There are multiple reports suggesting that patients with type 2 diabeteshave increased levels of plasma cortisol particularly in the periodbetween the nadir in the diurnal rhythm that occurs around midnight andthe early morning rise in cortisol. Cameron (Cameron et al. (1987).“Hypercortisolism in diabetes mellitus.” Diabetes Care 10(5): 662-4)reported that while the 24-hour cortisol levels were greater at all timepoints in patients with diabetes than non-diabetics, the largestdifference was at 8 am. This study also examined cortisol levels indiabetic patients following a dexamethasone suppression test. Followingingestion of 1 mg dexamethasone, cortisol levels remained significantlyelevated in the early morning in the diabetic patients but not in thecontrols. Similarly, night time (Lentle and Thomas (1964). “AdrenalFunction And The Complications Of Diabetes Mellitus.” Lancet 14: 544-9;Vakov (1984). “English translation of Circadian rhythm of cortisolsecretion in diabetes mellitus patients.”) and early morning (Lee et al.(1999). “Plasma insulin, growth hormone, cortisol, and central obesityamong young Chinese type 2 diabetic patients.” Diabetes Care 22(9):1450-7) cortisol levels were higher in patients with type 2 diabetesthan controls.

As cortisol will increase blood pressure and plasma glucose, therelationship between these parameters and cortisol in patients with type2 diabetes has been studied. One study reported that, in patients withtype 2 diabetes, there is a greater disturbance of the cortisol diurnalrhythm in those patients with hypertension compared to normotensivediabetics (Kostic and Secen (1997). “Circadian rhythm of blood pressureand daily hormonal variations” Med Pregl 50(1-2): 37-40). One studyreported that maturity onset, slightly overweight, non-insulin requiringdiabetic patients had higher cortisol levels than non-diabetics and thatdiabetic patients had a clear diurnal glucose rhythm and their peakglucose coincided with the peak cortisol (Faiman and Moorhouse (1967).“Diurnal variation in the levels of glucose and related substances inhealthy and diabetic subjects during starvation.” Clin Sci 32(1):111-26). Similarly, another study reported a strong correlation (r=0.82;p<0.01) between cortisol and glucose concentrations at 8:00 am inpatients with type 2 diabetes (Atiea et al. (1992). “The dawn phenomenonand diabetes control in treated NIDDM and IDDM patients.” Diabetes ResClin Pract 16(3): 183-90). One study found an increase in the 6 amcortisol levels in relatively lean type 2 diabetic patients and acorrelation (r=0.55; p<0.05) between plasma cortisol and the rate ofglucose production as measured by tracer dilution (Richardson and Tayek(2002). “Type 2 diabetic patients may have a mild form of an injuryresponse: a clinical research center study.” Am J Physiol EndocrinolMetab 282(6): E1286-90).

Adrenocorticotrophic hormone (ACTH, the pituitary hormone that regulatesadrenal corticosteroid production) has also been measured in a smallernumber of studies. One study examined cortisol and ACTH in normalvolunteers and in diabetes patients with and without autonomicneuropathy (AN). The diabetes patients with AN had higher HbA1c levelsthan the diabetic patients without AN and also had higher ACTH andcortisol levels than both the patients without AN and the controls(Tsigos et al. (1993). “Diabetic neuropathy is associated with increasedactivity of the hypothalamic-pituitary-adrenal axis.” J Clin EndocrinolMetab 76(3): 554-8). The increase in ACTH in the patients with diabetesand AN compared to the controls did not reach statistical significance.One study reported that ACTH was elevated in patients with type 2 (butnot type 1) diabetes (Vermes et al. (1985). “Increased plasma levels ofimmunoreactive beta-endorphin and corticotropin in non-insulin-dependentdiabetes.” Lancet 2(8457): 725-6).

In contrast to these predominantly positive correlative data, anotherstudy (Serio et al. (1968). “Plasma cortisol response to insulin andcircadian rhythm in diabetic subjects.” Diabetes 17(3): 124-6) reportednormal plasma levels of cortisol in patients diabetes. These patientshad quite mild diabetes as their glucose was controlled solely by diet.Similarly another study (with a smaller number of individuals) did notfind an increase in levels of circulating cortisol in patients with type2 diabetes (Kerstens et al. (2000). “Lack of relationship between11beta-hydroxysteroid dehydrogenase setpoint and insulin sensitivity inthe basal state and after 24 h of insulin infusion in healthy subjectsand type 2 diabetic patients.” Clin Endocrinol (Oxf) 52(4): 403-11).

Pharmacological intervention to reduce plasma cortisol has provedeffective in treating diabetes, hypertension and dyslipidemia inpatients with Cushing's syndrome. Sonino reported on 34 patients withCushing's syndrome who had their hypercortisolemia reduced byketoconazole at doses between 400 and 800 mg/day (Sonino et al. 1991supra). Three patients that were hyperglycemic but not on any diabetesmedications became euglycemic; of three other hyperglycemic patientsthat were on diabetes medications, one was able to discontinue the drugand the other two were able to reduce use of their hypoglycemicmedications. Similar results have been reported by Winquist (Winquist etal. 1995, “Ketoconazole in the management of paraneoplastic Cushing'ssyndrome secondary to ectopic adrenocorticotropin production.” J ClinOncol 13(1): 157-64). Ketoconazole also lowers blood pressure in themajority of patients with Cushing's syndrome (Sonino et al. 1991, supra;Fallo et al. (1993). “Response of hypertension to conventionalantihypertensive treatment and/or steroidogenesis inhibitors inCushing's syndrome.” J. Intern Med 234(6): 595-8).

Pharmacological reduction in cortisol synthesis has also been evaluatedin patients with type 2 diabetes. Metyrapone also inhibits 11βhydroxylase, the final step in the synthesis of cortisol and has beenused in short-term studies to determine whether acute suppression ofcortisol can have beneficial effects on glucose homeostasis. One study(Atiea et al. (1990). “Early morning hyperglycaemia “dawn phenomenon” innon-insulin dependent diabetes mellitus (NIDDM): effects of cortisolsuppression by metyrapone.” Diabetes Res 14(4): 181-5) used metyraponeto suppress the normal early morning rise in cortisol and reported thatthis intervention prevented the normal rise in glucose that occurs overthis time period. One study suppressed endogenous cortisol synthesis inpatients with type 1 diabetes using metyrapone and then infused cortisolto either mimic the normal nocturnal rise in cortisol or to produce alower basal level of cortisol. In the patients with a “suppressed”cortisol profile, there was a significantly lower rate of glucoseproduction (Dinneen et al. (1995). “Effects of the normal nocturnal risein cortisol on carbohydrate and fat metabolism in IDDM.” Am J Physiol268(4 Pt 1): E595-603). Carbenoxolone inhibits the activity of both HSD1and HSD2 and so lowers the exposure of liver and fat to cortisol.Another study treated both normal volunteers and patients with type 2diabetes for 7 days with carbenoxolone (Andrews et al. (2003). “Effectsof the 11 beta-hydroxysteroid dehydrogenase inhibitor carbenoxolone oninsulin sensitivity in men with type 2 diabetes.” J Clin EndocrinolMetab 88(1): 285-91). The patients with type 2 diabetes (but not thenormal volunteers) demonstrated a decrease in glucose production rateduring a euglycemic hyperinsulinemic hyperglucagonemic clamp. Racemicketoconazole has also been tested in patients with type 2 diabetes.These trials are consistent with the conclusion that therapeutic use ofthis drug to suppress cortisol synthesis can have beneficial effects onglucose, blood pressure and cholesterol in patients with type 2diabetes. While there may be an increase in cortisol levels or activityin patients with type 2 diabetes, therapeutic benefit can be obtained bya further reduction in cortisol levels or acitivity even in patientswith normal cortisol levels or activity.

While therapeutic use of racemic ketoconazole in patients with type 2diabetes has produced encouraging results, DIO-902 will be both moreefficacious and safer. DIO-902 has a significantly lower IC₅₀ toward thekey enzyme in cortisol synthesis (11β-hydroxylase) and a lower IC₅₀toward a key enzyme in cholesterol synthesis (14α-lanosteroldemethylase) than does the 2R,4S enantiomer (Rotstein et al. (1992).“Stereoisomers of ketoconazole: preparation and biological activity.” JMed Chem 35(15): 2818-25), thus potentially allowing a lower dose ofdrug to achieve the same efficacy. As demonstrated in Example 1, inrats, DIO-902 is more potent with respect to reducing corticosterone andcholesterol than is the 2R,4S enantiomer.

Furthermore DIO-902 has a 12× higher IC₅₀ toward CYP7A (IC₅₀=2.4 microM)than does the 2R,4S enantiomer (IC₅₀=0.195 microM) (Rotstein et al.1992, supra). Without intending to be bound by a particular mechanism,CYP7A suppression can lead to functional cholestasis and as aconsequence there can be hepatic and plasma accumulation of potentiallytoxic metabolites such as oxysterols and bilirubin and xenobiotics suchas ketoconazole itself. The reduced CYP7A inhibition associated withDIO-902 (compared to racemic ketoconazole) may account, at least inpart, for the unchanged toxicokinetics of DIO-902 observed afterrepeated dosing.

Preclinical studies have associated glucocorticoid activity with insulinresistance, hyperglycemia and increased adiposity, and clinical studiessupport the rationale for using cortisol synthesis inhibitors such asketoconazole as therapeutic options in patients with type 2 diabetes.Preclinical studies indicate that DIO-902 is safer and more efficaciousthan racemic ketoconazole.

C. Physical, Chemical, and Pharmaceutical Properties of an IllustrativePharmaceutical Formulation of the Invention—DIO 902

DIO-902 is the single enantiomer 2S,4R ketoconazole and is derived fromracemic ketoconazole. It is formulated using cellulose, lactose,cornstarch, colloidal silicon dioxide and magnesium stearate as animmediate release 200 mg strength tablet. The chemical name is 2S,4Rcis-1-acetyl-4-[4-[[2-(2,4-dichlorophenyl)-2-(1H-imidazol-1-ylmethyl)-1,3-dioxolan-4-yl]methoxyl]phenyl]piperazine,the formula is C₂₆H₂₈Cl₂N₄O₄, and the molecular weight is 531.44. TheCAS number is 65277-42-1, and the structural formula is provided below.The chiral centers are at the carbon atoms 2 and 4 as marked.

Ketoconazole is an imidazole-containing fungistatic compound. DIO-902 isan immediate release tablet to be taken orally and formulated as shownin the table below.

Component Percentage 2S,4R ketoconazole; 50% DIO-902 SilicifiedMicrocrystalline Cellulose, NF 16.5 (Prosolv HD 90) Lactose Monohydrate,NF (316 Fast-Flo) 22.4 Corn Starch, NF (STA-Rx) 10 Colloidal SiliconDioxide, NF (Cab-O- 0.5 Sil M5P) Magnesium Stearate, NF 0.6The drug product may be stored at room temperature and is anticipated tobe stable for at least 2 years at 25° C. and 50% RH. The drug ispackaged in blister packs.D. Non-Clinical Studies

1. Overview of Nonclinical Studies

This section contains pharmacology and toxicology information for bothDIO-902 and racemic ketoconazole. Pharmacology studies have includedstudies conducted to demonstrate the suppressive effects of DIO-902 oncorticosterone synthesis, serum cholesterol and testosterone levels inrats. The antifungal activity of DIO-902 has also been investigated inan in vitro study. The toxicology studies with DIO-902 in dogs includeda MTD study, a 7-day study, and a 28-day study (with toxicokinetics).Genotoxicity studies have also been conducted with DIO-902. BecauseDIO-902 is purified from racemic ketoconazole, the safety of the mixtureis relevant to that of DIO-902. Thus, this section includes a summary ofpharmacology and toxicology data taken primarily from the Summary Basisof Approval for NDA 18-533 for oral ketoconazole as well as data fromthe scientific literature and from a 28-day toxicity study in dogs.

2. Nonclinical Pharmacology

The primary pharmacological effect of DIO-902 will be through thesuppression of cortisol synthesis. Pharmacological intervention toreduce plasma cortisol has proved effective in treating diabetes,hypertension, and dyslipidemia in patents with Cushing's syndrome(Sónino et al. 1991, supra; Winquist et al. 1995, supra). Preclinicalstudies have associated glucocorticoid activity with insulin resistance,hyperglycemia, and increased adiposity (for a review see (McMahon et al.1988, supra). Secondary benefits of DIO-902 administration will includereduced cholesterol levels, reduced visceral adiposity, and reducedblood pressure.

A key enzymatic activity relevant to the therapeutic benefit of DIO-902is 11β hydroxylase, an enzyme that catalyzes the ultimate step inadrenal synthesis of cortisol. DIO-902 has been shown to inhibit thisenzyme with an IC₅₀ of 0.15 μM (see Table below). Because in rats themain glucocorticoid is corticosterone (in humans the main glucocorticoidis cortisol), the suppressive effects of DIO-902 on corticosteronesynthesis was investigated in rats. In one study, male Sprague Dawleyrats (10/group) received a single oral (via gastric tube) dose of 0, 50,100, 200, 400, and 600 mg/kg of 2S,4R-ketoconazole (DIO-902),2R,4S-ketoconazole, or racemic ketoconazole and were sacrificed 4 hourspost later. In another study, male Sprague Dawley rats (10/group)received a single oral (via gastric tube) dose of 0 or 200 mg/kg of2S,4R-ketoconazole (DIO-902), 2R,4S-ketoconazole, or the racemate andwere sacrificed at 4, 8, 12, 16, 20, and 24 hours post dosing. Theresults indicated that DIO-902 (the 2S,4R enantiomer) reduces plasmacorticosterone and does so more potently than the 2R,4S enantiomer, asshown in the following Tables. For more detail see Example 1.

Inhibition by DIO-902 of Enzymes that Catalyze Glucocorticol SynthesisIC₅₀ for IC₅₀ for 2S,4R IC₅₀ for Enzyme ketoconazole (DIO-902) 2R,4SReference 17α 0.91 NAV NAV (Ideyama et al. hydroxylase 1999*) 17, 20lyase 0.017 0.05 2.38 (Rotstein et al. 1992, supra; Ideyama et al. 1999)11β NAV 0.15 0.61 (Rotstein et al. 1992) hydroxylase aromatase NAV 11039.6 (Rotstein et al. 1992)All IC₅₀ values in the Table above are given in μM. While there may be asingle enzyme or complex responsible for both the 17β hydroxylase andthe 17,20 lyase activities, different IC₅₀ values have been reported forseveral inhibitors. NAV means data not available. * Ideyama et al.(1999). “YM116, 2-(1H-imidazol-4-ylmethyl)-9H-carbazole, decreasesadrenal androgen synthesis by inhibiting C17-20 lyase activity inNCI-H295 human adrenocortical carcinoma cells.” Jpn J Pharinaeol 79(2):213-20

In the following Table, the effect of ketoconazole enantiomers oncorticosteroid levels in rats is reported. In the Table, corticosteronelevels (mean±SEM; ng/mL) were determined four hours after oral gavage ofthe indicated drug (N=10/group). There was a single control group (dosedwith vehicle).

Effect of Ketoconazole Enantiomers on Corticosteroid Levels in RatsCorticosteroid Levels (mean ± SEM; ng/mL) Dose (mg/kg) 2S,4R (DIO-902)2R,4S Racemate  0 320 ± 9.4 320 ± 9.4 320 ± 9.4  50 186 ± 14.9 226 ±30.1 215 ± 20.7 100 139 ± 10.9 196 ± 17.2 210 ± 15.5 200 100 ± 8.6 217 ±25.8 135 ± 13.7 400  84 ± 11.6 192 ± 11.6 113 ± 6.6 600  80 ± 7.8 167 ±6.8 115 ± 14.3

The following Table presents the data from a study of the time course ofcorticosterone inhibition in rates following a single oral 200 mg/kgdose of ketoconazole enantiomers. Corticosterone levels (mean±SEM;ng/mL) were determined at the indicated times after oral gavage of theindicated drug at 200 mg/kg. So as to minimize the confounding effect ofthe diurnal corticosterone rhythm, all the rats were sacrificed at thesame time of day (1800 hours) and the time of drug administration wasdetermined to allow this (N=10/group). The means of the vehicle treatedgroups are used as the time zero control point.

Time Course of Corticosterone Inhibition in Rats Following a Single Oral200 mg/kg Dose Of Ketoconazole Enantiomers Corticosteroid Levels (mean ±SEM; ng/mL) Time (hours) Vehicle 2S,4R (DIO-902) 2R,4S Racemate  4 298 ±15.8  98 ± 10 191 ± 14 134 ± 10  8 374 ± 17 116 ± 13 206 ± 13 163 ± 1112 296 ± 21 113 ± 8 175 ± 9 153 ± 14 16 289 ± 16 133 ± 17 171 ± 9 132 ±6 20 323 ± 26 136 ± 12 169 ± 7 147 ± 17 24 341 ± 24 103 ± 14 182 ± 10151 ± 14

The secondary benefits of DIO-902 administration will include reducedLDL and total cholesterol, reduced visceral adiposity, reduced bloodpressure, and antifungal activity. The mechanism of action for DIO-902induced cholesterol suppression as well as pharmacology studiesdemonstrating the effects of DIO-902 on serum cholesterol andtestosterone levels in the rat are discussed below.

Racemic ketoconazole may directly lower cholesterol through inhibitionof lanosterol 14α demethylase activity, and the 2S,4R enantiomer has atwo fold lower IC₅₀ for this enzyme than does the other enantiomer(Rotstein et al. 1992, supra). The cholesterol lowering activity of the2S,4R enantiomer is expected to be further increased through diminishedinhibition of CYP7A, the principal enzyme controlling cholesterolcatabolism. Decreased CYP7A activity (in both humans (Pullinger et al.(2002). “Human cholesterol 7alpha-hydroxylase (CYP7A1) deficiency has ahypercholesterolemic phenotype.” J Clin Invest 110(1): 109-17) and inmice (Erickson et al. (2003). “Hypercholesterolemia and changes in lipidand bile acid metabolism in male and female cyp7A1-deficient mice.” JLipid Res 44(5): 1001-9) can lead to hypercholesterolemia, and so thesuppression of CYP7A by ketoconazole in humans (Pullinger et al. 2002,supra) is expected to attenuate the cholesterol lowering effect of thisdrug. The single enantiomer 2S,4R-ketoconazole is expected not to reduceCYP7A activity to the same extent as racemic ketoconazole. One studydemonstrated that the IC₅₀ of 2S,4R-ketoconazole (DIO-902) towards CYP7A(as measured by cholesterol 7α-hydroxylase activity) is 2.4 μM and theIC₅₀ of 2R,4S-ketoconazole is 0.195 μM, providing support that DIO-902has a 12× greater IC₅₀ toward CYP7A than 2R,4S-ketoconazole (Rotstein etal. 1992, supra).

A study was conducted to demonstrate the effect of DIO-902 oncholesterol levels in rats. In this study, male Sprague Dawley rats(10/group) received a single oral (via gastric tube) dose of 0 or 200mg/kg of 2S,4R-ketoconazole (DIO-902), 2R,4S-ketoconazole, or theracemate, and were sacrificed at 4, 8, 12, 16, 20, and 24 hours postdosing. The results, reported in the following Table, showed a smalldecrease in cholesterol levels at 16, 20 and 24 hours after treatmentwith the 2S,4R enantiomer but not with the racemate or with the otherenantiomer. Cholesterol levels (mean±SEM; mg/dL) were determined at theindicated times after oral gavage of the indicated drug at 200 mg/kg.All rats were sacrificed at the same time of day (1800 hours) and thetime of drug administration was determined appropriately (N=10/group).

Effect of Ketoconazole Enantiomers on Serum Cholesterol in RatsCholesterol levels (mean ± SEM; mg/dL) Time 2S,4R (DIO- (hours) Vehicle902) 2R,4S Racemate  4 77.3 ± 3.9 69.6 ± 1.9 85.1 ± 7 81.2 ± 3.9  8 73.5± .5 73.5 ± 3.1 85.1 ± 5.4 73.5 ± 2.3 12 69.6 ± 3.5 77.3 ± 3.9 77.3 ±1.9 69.6 ± 2.3 16 69.6 ± 1.9 61.9 ± 3.1 77.3 ± 4.6 69.6 ± 3.1 20 69.6 ±1.9   58 ± 1.2 69.6 ± 2.7 65.7 ± 2.7 24 65.7 ± 2.7 61.9 ± 3.1 69.6 ± 1.565.7 ± 3.9

Two studies were conducted to investigate the effect of DIO-902 ontestosterone levels in rats. In one study, male Sprague Dawley rats(10/group) received a single oral (via gastric tube) dose of 0, 50, 100,200, 400, and 600 mg/kg of 2S,4R-ketoconazole (DIO-902),2R,4S-ketoconazole, or racemic ketoconazole, and were sacrificed 4 hourspost dosing. In another study, male Sprague Dawley rats (10/group)received a single oral (via gastric tube) dose of 0 or 200 mg/kg of2S,4R-ketoconazole (DIO-902), 2R,4S-ketoconazole, or the racemate, andwere sacrificed at 4, 8, 12, 16, 20, and 24 hours post dosing. Theresults shown in the following Tables demonstrate that the 2S,4Renantiomer (DIO-902) is more potent at suppressing testosterone than isthe other enantiomer (2R,4S). For the results shown in the immediatelyfollowing Table, testosterone levels (mean±SEM; nmol/L) were determinedfour hours after oral gavage of the indicated drug (N=10/group). Therewas a single control group (dosed with vehicle).

Effect of Ketoconazole Enantiomers on Testosterone in Rats DoseTestosterone levels (mean ± SEM; nmol/L) (mg/kg) 2S,4R (DIO-902) 2R,4SRacemate  0 2.7 ± 0.5 2.7 ± 0.5 2.7 ± 0.5  50 2.6 ± 0.7 2.5 ± 0.5 2.7 ±0.6 100 0.8 ± 0.3 1.3 ± 0.2 1.7 ± 0.5 200 0.2 ± 0.1 1.4 ± 0.4 0.4 ± 0.2400 0.3 ± 0.1 0.7 ± 0.2 0.4 ± 0.2 600   0 ± 0 1.6 ± 0.3 0.8 ± 0.1

For the results shown in the following Table, testosterone levels(mean±SEM; nmol/L) were determined at the indicated times after oralgavage of the indicated drug at 200 mg/kg. All rats were sacrificed atthe same time of day (1800 hours), and the time of drug administrationwas determined appropriately (N=10/group). The means of the vehicletreated groups are used as the time zero control point. Although 2S,4Ris more potent than 2R,4S with respect to acute suppression oftestosterone, the overall physiological consequences may be reduced with2S,4R as opposed to 2R,4S. As noted in Example 2, the concentration ofthe 2S,4R enantiomer does not increase with repeated doses. This iscontrast to the concentration of the racemic mixture, which doesincrease with repeated doses. Thus, with repeated doses of the racemicmixture, testosterone suppression will become more marked with time. Asalso noted in Example 3, the concentration of the racemic mixture 24hours after taking the drug increases markedly between the first andsubsequent doses. Thus testosterone suppression will last progressivelylonger during the day in the inter-drug interval. As the 2S,4Renantiomer does not inhibit its own clearance, the period during the daywhen testosterone production is suppressed will not get progressivelylonger.

Time Course of Testosterone Suppression Following a Single Dose (200mg/kg) of Ketoconazole Enantiomers in Rats Testosterone levels (mean ±SEM; nmol/L) Time 2S,4R (DIO- (hours) Vehicle 902) 2R,4S Racemate  4 3.7± 0.7 0.8 ± 0.2 3.4 ± 0.5 1.6 ± 0.6  8 8.9 ± 1.4 5.9 ± 0.8 8.6 ± 2.0 8.0± 1.0 12 5.4 ± 1.1 3.4 ± 0.5 5.6 ± 1.1 4.6 ± 0.6 16 5.6 ± 0.9 3.9 ± 0.65.5 ± 0.9 3.8 ± 0.5 20 5.7 ± 1.0 5.2 ± 0.6 5.2 ± 1.1 5.6 ± 0.8 24 5.5 ±0.9 4.4 ± 0.7 6.0 ± 1.0 5.9 ± 0.5

3. Antifungal Activity.

In an in vitro study, both DIO-902 and 2R,4S-ketoconazole exhibitantifungal activity as reported in the following Table. In this study,yeast isolates were incubated with racemic ketoconazole, DIO-902(2S,4R-ketoconazole), 2R,4S-ketoconazole, or solvent (DMSO) for 48 hoursat 36±1° C., and the minimum inhibitory concentration (MIC) wasdetermined. The MIC was defined as the lowest concentration thatsubstantially inhibited growth of the organism (i.e. that caused aprominent decrease of greater than or equal to 80% in turbidity comparedto that of controls).

Antifungal Activity of DIO-902 DSM MIC (mg/L) Strain 2S,4R NumberOrganism Ketoconazole 2R,4S (DIO-902) 11948 Candida albicans <0.015<0.015 <0.015 11944 Candida albicans <0.015 <0.015 <0.015 11949 Candidaalbicans 0.125 0.25 0.125 11945 Candida albicans 0.03 0.03 0.03 11943Candida albicans <0.015 <0.015 <0.015 11225 Candida albicans <0.015<0.015 <0.015 98-St- Candida albicans <0.015 <0.015 <0.015 00799 11950Candida glabrata 0.25 0.25 0.25 11226 Candida glabrata 0.5 0.5 0.5 11947Candida 0.125 0.25 0.06 guilliermondii 11954 Candida kefyr 0.03 0.030.03 05784 Candida parapsilosis 0.03 0.06 0.03 11224 Candidaparapsilosis <0.015 0.06 0.03 11952 Candida tropicalis 4 4 2 11953Candida tropicalis 0.06 0.125 0.06 11951 Candida tropicalis 0.125 0.1250.06 11960 Cryptococcus 0.125 0.125 0.125 neoformans 11959 Cryptococcus0.06 0.03 0.03 neoformans 11956 Issachenkia orientalis 0.25 0.5 0.2511958 Issatchenkia orientalis 1 2 1 01333 Saccharomyces 0.25 0.25 0.25cerevisiae

While the anti-fungal activity of the 2S,4R enantiomer has been assertedwithout proof, these results demonstrate for the first time that thisenantiomer is surprisingly more effective as an anti-fungal agent thanthe racemate and/or 2R,4S enantiomer against a variety of fungi,including Issatchenkia orientalis, Issachenkia orientalis, Cryptococcusneoformans, Candida tropicalis, Candida parapsilosis, Candidaguilliermondii, and Candida albicans, or certain strains thereof. In oneembodiment, the present invention provides a method for treating afungal infection of one of these fungi or strains of fungi byadministering a therapeutically effective amount of a pharmaceuticalcomposition of the 2S,4R enantiomer of ketoconazole substantially freeof the 2R,4S enantiomer.

4. Safety Pharmacology

The inhibitory potential of DIO-902 on CYP3A inhibitory activity hasbeen studied. In this study, DIO-902 and the 2R,4S ketoconazoleenantiomer were shown to have IC₅₀ values that were comparable to eachother and to the racemic mixture although there was be a small (2×)increase in the IC₅₀ of the 2S,4R-enantiomer toward CYP3A5. DIO-902(0.005-50 μM for CYP3A4 and 0.01-100 μM for CYP3A5) was added tomicrosomes prepared from human liver or to recombinant 3A4 and 3A5. As apositive control and as a comparator, the activities of the otherenantiomer (2R,4S) and the racemic mixture were also determined. Thesubstrate used in these experiments was quinone, an establishedsubstrate for the CYP3A4 and CYP3A5 (Mirghani et al. (2002). “Enzymekinetics for the formation of 3-hydroxyquinine and three new metabolitesof quinine in vitro; 3-hydroxylation by CYP3A4 is indeed the majormetabolic pathway.” Drug Metab Dispos 30(12): 1368-71).

Activity of DIO-902 Towards the Hydroxylation of Quinine HLM poolrCYP3A4 rCYP3A5 Quinine 160 μM Quinine 30 μM Quinine 20 μM IC₅₀ μM IC₅₀μM IC₅₀ μM racemate 0.27 0.12 0.38 2R,4S 0.37 0.14 0.40 2S,4R 0.29 0.100.71 (DIO-902) HLM: human liver microsomes

The scientific literature reports the inhibitory activity of the 2S,4Renantiomer on Cytochome P450 Inhibition. One study (Rotstein et al.1992, supra) evaluated the inhibitory activity of the two ketoconazoleenantiomers (2S,4R and 2R,4S ketoconazole) toward the hydroxylation ofprogesterone, lauric acid, and cholesterol which are markers for variousP450 enzymes. The IC₅₀ of the 2S,4R enantiomer was slightly greater thanthat of 2R,4S. The IC₅₀ for the inhibition of CYP3A4 (via6β-hydroxylase) was similar to that of racemic ketoconazole as reportedby Swinney, 1990. Specifically, the IC₅₀ for the inhibition ofprogesterone 6β-hydroxylase metabolism in rat hepatic microsomes was 1.4μM. Due to the similar IC₅₀ for CYP450 3A4 inhibition for the 2S,4Renantiomer and racemic ketoconazole, the potential for drug metabolisminteractions for these two compounds is expected to be similar. Howeveras noted below and in Example 2, the potential for DIO-902 to cause achange in PK profile of an administered drug through an inhibition ofdrug excretion is signicantly reduced compared to that of the otherenantiomer.

In regards to activity of the P450 enzyme, CYP7A (cholesterol 7αhydroxylase), the results, shown in the Table below, demonstrate thatthe IC₅₀ of the 2S,4R enantiomer is approximately 12-fold higher thanthe IC₅₀ of the 2R,4S enantiomer. CYP7A is relevant to the issue of druginteraction, because this enzyme controls bile formation, and thus, theexposure to drugs that are normally cleared via the bile may be alteredunder conditions of reduced bile formation and flow. It has been shownthat racemic ketoconazole inhibits bile formation through inhibition ofCYP7A. Racemic ketoconazole has been shown to reduce bile flow and theclearance of endogenous metabolites (cholesterol) and xenobiotics(bromosulphopthalein) into the bile (Princen et al. (1986).“Ketoconazole blocks bile acid synthesis in hepatocyte monolayercultures and in vivo in rat by inhibiting cholesterol 7alpha-hydroxylase.” J Clin Invest 78(4): 1064-71; Gaeta and Tripodi(1987). “Ketoconazole impairs biliary excretory function in the isolatedperfused rat liver.” Naunyn Schmiedebergs Arch Pharmacol 335(6):697-700). That the 2S,4R enantiomer has a reduced impact on thepharmacokinetics of a drug (ketoconazole) that is normally cleared viathe bile may due to the observation that the IC₅₀ of the 2S,4Renantiomer is approximately 12-fold higher than the IC₅₀ of the 2R,4Senantiomer toward CYP7A. As a consequence of this reduced inhibition ofdrug clearance, the 2S,4R enantiomer will significantly decrease therisk of hepatic damage as compared to the other enantiomer or to theracemic mixture of the two enantiomers that constitute ketoconazole.

P450 inhibitory activity of the ketoconazole enantiomers (Rotstein etal. 1992, supra) IC₅₀ (μM) Associated 2S,4R (DIO- Substrate ReactionP450 902) 2R,4S Progesterone 2α hydroxylase 2C11 104 84 Progesterone 6βhydroxylase 3A 1.3 0.79 Progesterone 16α 2B1, 2B2, 84 69 hydroxylase1A1, 2C11, 3A Progesterone 21 hydroxylase 2C6 9.0 11.2 Lauric acidhydroxylase 4A >100 <100 Cholesterol 7α hydroxylase 7A 2.4 0.195

5. Nonclinical Pharmacokinetics

The absorption of DIO-902 (2S,4R enantiomer) was studied during a 28 daydog toxicology study. In this study, dogs were treated orally withDIO-902 doses of 2, 6.5, and 20 mg/kg. Serum samples were taken afterthe 1^(th) and 28^(th) daily dose of the 2S,4R enantiomer. Forcomparison, a group of dogs were to receive racemic ketoconazole at adose of 40 mg/kg/day for 28 days. This dose was administered as plannedfor the first 9 days of the study; however, due to toxicity, the 40mg/kg dose was discontinued after the 9^(th) day, and animals in thisgroup were left untreated for the next 5 days (days 10 to 14). Beginningon study day 15 and continuing through study day 28, animals weretreated with 20 mg/kg of ketoconazole. Toxicokinetic parameters aresummarized in the Tables below.

The plasma levels in the dogs dosed with DIO-902 at 2 mg/kg/day werebelow detection for most of the 24 hour profile. Thus, an accurate AUCcould not be calculated for this dose. Where AUC was calculated, it wasbased on the values that were above the limit of detection over the timeperiod from 0 to 12 hours post dosing (see Table below). As such, theAUC from the 2 mg/kg dose cannot reliably be compared with that of theother dose levels. The AUC and C_(max) values at 2, 6.5, and 20 mg/kgwere comparable between Day 1 and Day 28 for each DIO-902 dose level,indicating minimal to no accumulation with repeat dosing. No sexdifferences were seen in DIO-902 treated animals. C_(max) and AUC levelsin animals treated with 2 mg/kg or 6.5 mg/kg DIO-902 were approximatelyproportional to dose. At the 6.5 and 20 mg/kg dose levels, the increasein AUC and C_(max) levels were increased more than that of the increasein dose. T_(max) values ranged from 1 to 8 hours on Day 1 and 1 to 12hours on Day 28 (see the second of the two following Tables).

For racemic ketoconazole, the AUC and plasma drug levels on Day 28 werenotably lower than that seen on Day 1 due to the interruption in dosingand the reduced dose levels administered. However, both the AUC andC_(max) values are decreased more than the decrease in dose from Day 1to Day 28. Thus, Day 1 and Day 28 data for racemic ketoconazole cannotbe reliably compared. When comparing the Day 1 data for the ketoconazole40 mg/kg dose with that of the 20 mg/kg dose for DIO-902, the AUC andC_(max) values in the animals treated with racemic ketoconazole areapproximately double that of the animals treated with 20 mg/kg ofDIO-902. On Day 28, the AUC and C_(max) values from the animals treatedwith 20 mg/kg of racemic ketoconazole were substantially lower than thatof animals treated with 20 mg/kg of DIO-902.

Due to the issues discussed above with the doses of racemicketoconazole, for comparison purposes, additional data for racemicketoconazole from another 28-day toxicity study in dogs was obtained. Inthis study, dogs (3/sex/group) were treated with oral doses of 2.5, 10,or 40 mg/kg of racemic ketoconazole in a powder suspension or 2.5, 10 or40 mg/kg of racemic ketoconazole in an oil suspension once daily for 28days. Toxicokinetic samples were collected on Day 1 and during Weeks 2and 4. For comparison with the current data, Day 1 and Day 28 data arepresented from the administered ketoconazole powder suspension (10 and40 mg/kg). Data from the oil suspension was similar to the powdersuspension. The C_(max) values for DIO-902 on day 28 for dogs dosed at20 mg/kg/day were between 9.94 microg/ml and 9.95 microg/ml (see thesecond of the two following Tables). For comparison, a dose of 10 mg/kgof racemic ketoconazole produced a C_(max) of 7.52 to 9.20 μg/ml (on day28) and a dose of 40 mg/kg led to a C_(max) of 42.78 to 46.75 μg/ml (onday 28). In contrast to that seen with racemic ketoconazole, it isapparent that the AUC and C_(max) for 2S,4R ketoconazole (DIO-902) werenot significantly different on day 28 as compared to day 1. Asignificant increase between day 1 and day 28 was noted for racemicketoconazole (see the second of the two following Tables). For thefollowing Table: *Days of treatment. The limit of detection was 0.25μg/ml. a: Data for racemic ketoconazole. b: Data for racemicketoconazole. Values represent mean of 3 animals.

Plasma drug levels of DIO-902 and Racemic Ketoconazole in Dogs followingSingle and Repeat Oral Dosing Dose Day Drug concentration (μg/ml) at theindicated time (hours) Drug (mg/kg) * Sex 0 1 2 4 8 12 DIO-902 2 1 M<0.25 0.40 0.45 <0.25 <0.25 <0.25 F <0.25 <0.25 0.27 0.28 <0.25 <0.25 228 M <0.25 0.66 0.54 0.38 <0.25 <0.25 F <0.25 0.29 0.52 0.30 <0.25 <0.256.5 1 M <0.25 1.19 1.62 1.25 0.41 0.40 F <0.25 <0.25 0.44 2.32 0.50<0.25 6.5 28 M <0.25 1.17 1.39 1.54 1.33 0.88 F <0.25 0.25 1.25 1.851.27 0.34 20 1 M <0.25 7.05 8.30 6.15 2.92 6.74 F <0.25 <0.25 0.65 9.729.95 5.44 20 28 M 1.19 9.13 9.78 8.17 5.86 4.25 F 2.88 1.78 2.43 6.429.83 6.53 Racemic 20 28 M <0.25 <0.25 0.28 <0.25 <0.25 <0.25ketoconazole^(a) F <0.25 <0.25 0.27 0.35 0.31 <0.25 40 1 M <0.25 5.608.87 10.82 16.63 12.76 F <0.25 4.29 12.33 20.09 18.33 14.82 Racemic 10 1M <0.25 0.38 0.62 1.18 0.33 <0.25 ketoconazole^(b) F <0.25 1.30 1.230.59 <0.25 <0.25 10 28 M <0.25 7.53 8.63 6.20 1.44 0.43 F <0.25 7.287.21 4.39 0.85 <0.25 40 1 M <0.25 10.30 14.60 23.09 10.12 6.70 F <0.255.65 5.76 3.30 1.84 1.55 40 28 M 3.97 11.90 24.63 32.72 46.75 28.29 F12.84 12.28 31.55 38.59 40.45 29.91

Toxicokinetics of DIO-902 in Dogs following Single and Repeat OralDosing Dose Days of AUC₍₀₋₁₂₎ Cmax Tmax Range Drug (mg/kg) treatment Sex(μg · h/mL) (μg/mL) (hour) DIO-902 2 1 M 2.39* 0.51 1-2 F 2.57* 0.45 2-42 28 M 3.26 0.68 1-4 F 2.76* 0.75 1-8 6.5 1 M 12.23** 2.00 1-4 F 4.70*2.57 2-4 6.5 28 M 14.65 2.01 1-8 F 12.86 2.41 2-8 20 1 M 60.59 9.38 1-4F 80.63** 14.66 4-8 20 28 M 80.00 9.94 2-4 F 77.85 9.95  8-12 Racemic 401 M 142.80 16.63 8 ketoconazole^(a) F 185.41 21.28 4-8 20 28 M 3.42*0.51 1-8 F 4.70* 0.55 2-8 Racemic 10 1 M 9.01 1.43 2-4 ketoconazole^(b)F 6.73 1.58 1-8 10 28 M 44.15 9.20 1-2 F 33.11 7.52 1-2 40 1 M 179.8223.32 2-4 F 42.94 6.23  2-12 M 542.28 46.75 8 F 639.19 42.78 4-8 For thepreceding Table, the data provided in the first of the two precedingTables were used to derive AUC and Cmax values on the first day ofdosing and again after 28 daily days of dosing. Values represent mean of3 animals. *n = 1, **n = 2. ^(a):Data for racemic ketoconazole.^(b):Data for racemic ketoconazole. AUC data is for 0-24 h.

6. Repeat Dose Toxicity of DIO-902

The toxicity of DIO-902 has been investigated in dogs in a maximumtolerated dose study, a 7-day study, and a 28-day study in dogs. The MTDinvestigation and 7-day study were conducted as separate phases of asingle study.

In a GLP maximum tolerated dose study, Beagle dogs (2/sex) were treatedorally (capsule) with ascending doses (20, 40, 60 and 80 mg/kg) of the2S,4R enantiomer. As a control, a separate set of 2 male and 2 femaledogs was treated with vehicle. Animals were treated with each dose forthree days before ascending to the next higher dose. There were nodeaths during the ascending phase. Clinical signs were noted at 40 mg/kg(vomiting). At higher doses head shaking, tremors, salivation, coloredurine and liquid feces were noted. The 80 mg/kg dose was abandoned onwelfare grounds. Food intake and weight gain was reduced at all doses.

After the end of the MTD study, the 4 animals that were treated withvehicle were treated orally (capsule) with 40 mg/kg of the enantiomerfor 7 days. No control group was included. All animals survived toscheduled sacrifice. During the fixed dose (7 days at 40 mg/kg/day), onedog was noted as being thin, and one dog was noted as having tears.There were no post-dosing observations. Food consumption by all fouranimals was reduced and all four lost weight over the seven day studyperiod. Hematological analysis suggested a decrease in reticulocytes(absolute and relative) in one dog and a 20% reduction in total whitecell numbers. The mean ALT levels in the treated dogs increased by lessthan two fold compared to the mean determined prior to dosing. There wasno significant change in any of the other liver enzymes measures.Macroscopic findings at necropsy were limited to areas of GI irritation.There may have been an increase in the weights of liver and kidney, butin the absence of a concurrent control, this could not be concluded withconfidence.

In a 28 day GLP study, beagle dogs (3/sex/group) received daily oral2S,4R enantiomer doses of doses of 0 (placebo), 2, 6.5, or 20 mg/kg. Aseparate control group (3/sex) was included and treated orally withracemic ketoconazole at an initial dose of 40 mg/kg/day. At 40 mg/kg ofracemic ketoconazole, significant body weight losses (up to 17.3%) ledto cessation of dosing after 9 days. The dogs in this group (3/sex) weretaken off drug for 6 days and then restarted at 20 mg/kg/day. Thetoxicokinetic profile taken at 28 days indicated that the C_(max) ofracemic ketoconazole on day 28 was less than 5% of that determined onday 1. Thus, for data comparison, this group cannot be used withconfidence as a comparator. Unless otherwise noted below, all furtherreferences to drugs and doses in this study will refer to the singleenantiomer 2S,4R.

Toxicokinetic data indicated that DIO-902 was systemically absorbed. Ata 2 mg/kg DIO-902 dose level, the plasma drug levels were below thelimit of detection at many of the timepoints between 1 and 12 hours postdosing. Thus, AUC was calculated using data from timepoints were plasmadrug levels were above the limit of detection. For each dose, no sexdifferences were observed and no accumulation occurred over the 28 daysof dosing.

The dogs dosed with DIO-902 at 20 mg/kg/day ate approximately 25-35%less food than those in the placebo control group. The dogs dosed at 20mg/kg/day gained 0.25 kg (males) and 0.14 kg (females) compared to theplacebo treated dogs that gained 1.1 kg (males) and 0.9 kg (females) inbody weight. The trends indicate that most of the effects on body weightwere in the first two weeks of the study and that at the end of thestudy the dogs dosed at 20 mg/kg/day were gaining weight at a ratesimilar to the placebo control group. Food intake also increased in the20 mg/kg/day group although still below the placebo control group. Atthe intermediate doses there were no obvious effects on food intake orweight gain.

There were no measurable effects of DIO-902 at these doses on any of theophthalmological or electrocardiographic parameters that were measured.Specifically in the dogs treated with DIO-902 at 20 mg/kg/day, there wasno obvious QTc prolongation. There were no hematological changes noted.There was no change in the urinalysis. The only change in any serumchemistry measures was a reduction in cholesterol. There were trends ofdecrease in the kidney weights in the female dogs and trends of anincrease in the relative (but not absolute) weights of the liver andadrenals in male and females. There were no remarkable microscopicfindings at any dose.

7. Other Toxicity Testing

No reproductive toxicology studies have been conducted with DIO-902;however, the reproductive toxicity of racemic ketoconazole has beenextensively investigated studied.

DIO-902 was found to be negative for genotoxicity in an Ames assay andin the mouse lymphoma assay. In the Ames assay, DIO-902 was assayed withrespect to mutation induction in five different histidine requiringstrains of Salmonella typhimurium. Exposure to the DIO-902 produced nodose related and repeatable increase in revertant numbers. In thelymphoma assay, DIO-902 (with and without S-9 activation) was studiedwith respect to the induction of mutations at the thymidine kinase locusin mouse L5178Y lymphoma cells. DIO-902 did not reproducibly ormeaningfully induce mutation at the TK locus in three independentexperiments in the absence of S-9 and two independent experiments in thepresence of S-9 when tested up to toxic doses.

Carcinogenicity studies have not been conducted with DIO-902. Racemicketoconazole has been found to be non-carcinogenic (SBA for NDA 18-533).

Administration of the 2S,4R enantiomer substantially free of the 2R,4Senantiomer is expected to reduce the risk of hepatic reactions sometimesseen following administration of racemic ketoconazole (Stricker et al.(1986). “Ketoconazole-associated hepatic injury. A clinicopathologicalstudy of 55 cases.” J Hepatol 3(3): 399-406; Lake-Bakaar et al. (1987).“Hepatic reactions associated with ketoconazole in the United Kingdom.”Br Med J (Clin Res Ed) 294(6569): 419-22; Van Cauteren et al. (1990).“Safety aspects of oral antifungal agents.” Br J Clin Pract Suppl 71:47-9; and Rodriguez and Acosta (1997). “Metabolism of ketoconazole anddeacetylated ketoconazole by rat hepatic microsomes andflavin-containing monooxygenases.” Drug Metab Dispos 25(6): 772-7).Ketoconazole induced hepatic reactions are usually described asidiosyncratic (Stricker et al. 1986, supra) implying that the underlyingmechanism(s) are not known. It has been demonstrated that racemicketoconazole inhibits bile formation in rats through inhibition of CYP7A(Princen et al. 1986, supra). Racemic ketoconazole has been shown toinhibit human CYP7A (Rotstein et al. 1992, supra), reduce bile acidsynthesis by human hepatocytes (Princen et al. 1986, supra), and inhibitbile acid production (Miettinen 1988, supra) in treated patients. Webelieve that a key component of ketoconazole induced hepatotoxicity isthe inhibition of CYP7A. Because DIO-902 has a 12× higher IC₅₀ towardCYP7A (IC₅₀=2.4 μM) than does the other enantiomer 2R,4S (IC₅₀=0.195 μM)and does not undergo the time dependent increase in drug concentrationseen for the racemate, DIO-902 will be associated with a significantlylower incidence of liver reactions. The two effects should beinteractive; that is, the racemate will accumulate more than DIO-902,and the higher drug accumulation of the racemate will lead to an evengreater relative inhibitory effect on CYP7A than is implied from thecell free assays. The relevant drug concentrations attained in humans,the relative levels in plasma of the two enantiomers, and the relativeIC₅₀ values are consistent with this expectation.

E. Pharmacokinetics of DIO-902 in Humans

No clinical trials have yet been conducted with DIO-902. However thepharmacokinetic profile of the individual enantiomers following thefirst and the fifth 200 mg dose of racemic ketoconazole (doses givenevery twelve hours) have been presented in poster form (Gerber (2003).“Stereoselective pharmacokinetics (PK) of oral ketoconazole (K) inhealthy subjects.” ACAAF poster). The pharmacokinetic data aresummarized in the following Table. The exposure to DIO-902, 2S,4Renantiomer, is approximately 2.5 fold that of the 2R,4S enantiomer. Itis not clear if this results from a difference in bioavailability orclearance. After five doses, the AUC and the Cmax increase for bothenantiomers. As exposure to the 2R,4S enantiomer could alter theclearance of both 2S,4R and 2R,4S enantiomers, this result is notnecessarily at variance with the pharmacokinetic data obtained frompreclinical results obtained in dogs dosed with DIO-902, the singleenantiomer. Pharmacokinetic Data (Gerber 2003, supra)

Following first dose Following fifth dose DIO-902; DIO-902; 2S,4R 2R,4S2S,4R 2R,4S AUC 0-12  302 +/− 38  820 +/− 142  538 +/− 74  1543 +/− 231(μg*min/mL) T ½  133 +/− 14   97 +/− 8  217 +/− 30   158 +/− 19(minutes) C_(max) μg/mL 1.06 +/− 0.13  3.4 +/− 0.44 1.53 +/− 0.19  4.77+/− 0.55F. Idiosyncratic Liver Reactions in Humans

The idiosyncratic liver reactions to racemic ketoconazole have beendescribed (Stricker et al. 1986, supra). The description of theseresponses as being idiosyncratic implies that there is no clearunderstanding of the mechanism(s). Any coherent mechanistic explanationshould encompass the asymptomatic increase in liver enzymes that occurswithin a short period of time in 1-10% of treated patients followingfirst exposure, as well as the relatively infrequent incidence of moresevere responses. There is no consistent evidence linking ketoconazoleto immune mediated mechanisms.

Although no relationship between dose and hepatotoxicity in humans hasbeen described, there is a clear correlation between AUC and liverdamage in rabbits (Ma et al. (2003). “Hepatotoxicity and toxicokineticsof ketoconazole in rabbits.” Acta Pharmacol Sin 24(8): 778-82). Theseauthors reported that, in rabbits, 40 mg/kg ketoconazole inducedmorphological changes in hepatocytes and an increase in serum liverenzymes. This dose is comparable to the highest dose tested in a oneyear dog study. Acute in vitro hepatoxicity was studied by others(Rodriguez and Acosta 1997, supra, and Rodriguez and Acosta (1997).“N-deacetyl ketoconazole-induced hepatotoxicity in a primary culturesystem of rat hepatocytes.” Toxicology 117(2-3): 123-31). In thesestudies, rat hepatocytes were cultured in the presence of increasingdoses of ketoconazole (up to 200 microM) for times that ranged from 0.5hour to 4 hours. These authors found that there was both a dose and timecomponent to the release of lactate dehydrogenase (LDH). At the longesttime exposure studied (four hours), there was no detectable effect ofketoconazole at concentrations below 75 microM (39 μg/mL). There is alsoa suggestion from preclinical animal models that the metabolites ofketoconazole (specifically deacetylated ketoconazole (DAK)) is a morepotent mitochondrial inhibitor than ketoconazole (Rodriquez and Acosta(1996). “Inhibition of mitochondrial function in isolated rat livermitochondria by azole antifungals.” J Biochem Toxicol 11(3): 127-31).The in vitro IC₅₀ for DAK inhibition of succinate dehydrogenase is 350microM (in comparison to the C_(max) of unmetabolized ketoconazole of12.3 microM following a 400 mg dose in humans (Huang et al. (1986).“Pharmacokinetics and close proportionality of ketoconazole in normalvolunteers.” Antimicrob Agents Chemother 30(2): 206-10) It is possiblethat these and related direct effects of ketoconazole (and themetabolites) could lead to an idiosyncratic reaction if there werepatients that were significantly more susceptible than the generalpopulation.

Material provided here and in Example 2 indicate that a key component ofketoconazole induced hepatotoxicity is the inhibition of CYP7A. BecauseDIO-902 has a 12× higher IC₅₀ toward CYP7A (IC₅₀=2.4 microM) than doesthe other enantiomer 2R,4S (IC₅₀=0.195 microM) (Rotstein et al. 1992,supra) and does not undergo the time dependent increase in drugconcentration seen for the racemate, DIO-902 will be associated with asignificantly lower incidence of liver reactions. As noted above, thetwo effects will be interactive; that is the racemate will accumulatemore than DIO-902 and the higher drug accumulation of the racemate willlead to an even greater relative inhibitory effect on CYP7A than isimplied from the cell free assays. The inhibition of CYP7A by racemicketoconazole may cause a hepatic reaction indirectly through reducedbile acid synthesis and the consequent reduction in bile flow andincrease in potentially toxic metabolites. Ketoconazole may furtherexacerbate this process by directly increasing the level of potentiallyhepatotoxic oxysterols.

Racemic ketoconazole inhibits bile formation in rats through inhibitionof CYP7A (Princen et al. 1986, supra) (bile synthesis is blocked whencholesterol is used as a substrate but not when 7α-cholesterol is usedas a substrate). The inhibition of bile acid synthesis by ketoconazoleis a direct effect on hepatocytes (Whiting et al. (1989). “Bile acidsynthesis and secretion by rabbit hepatocytes in primary monolayerculture: comparison with rat hepatocytes.” Biochim Biophys Acta 1001(2):176-84). Bile flow is also reduced by ketoconazole and the clearance ofendogenous metabolites (cholesterol) (Princen et al. 1986, supra) andxenobiotics ((bromosulphopthalein (Gaeta and Tripodi 1987, supra)) intothe bile is reduced. As ketoconazole is excreted into the bile, it wouldbe anticipated that ketoconazole might inhibit its own clearance andlead to increased plasma concentrations. This increase in drugconcentration has been noted in humans and in dogs. That CYP7Ainhibition causes functional cholestasis (reduced bile acid synthesisand bile flow) is consistent with the recognition that CYP7A is the ratelimiting step in bile acid synthesis, and bile acid synthesis appears tobe rate limiting for bile flow. In humans, the genetic absence offunctional CYP7A causes a profound decrease in fecal bile acids(Pullinger et al. 2002, supra) and in mice, the genetic absence of CYP7Acan cause cholestasis (Amon et al. (1998). “Cholesterol 7-hydroxylaseknockout mouse: a model for monohydroxy bile acid-related neonatalcholestasis.” Gastroenterology 115(5): 1223-8).

The relationship between CYP7A inhibition, cholestasis, and liver damageis also consistent with other rodent models that do not use ketoconazoleas an experimental tool. Thus, ethinylestradiol induced cholestasis inrats correlates with a suppression of bile flow, liver bile acidcontent, and liver cholesterol content. Epomediol preventsethinylestradiol induced cholestasis and produces significant (albeitsmall) reversals in these three measures. CYP7A activity was suppressedby ethinylestradiol and returned to normal with epomediol (Cuevas et al.(2001). “Effect of epomediol on ethinyloestradiol-induced changes inbile acid and cholesterol metabolism in rats.” Clin Exp PharmacolPhysiol 28(8): 637-42). Ketoconazole inhibits human microsomal CYP7A,reduces bile acid synthesis by human hepatocytes (Princen et al. 1986,supra) and inhibits bile acid production (Miettinen 1988, supra) intreated patients. Functional cholestasis can cause subsequent hepaticdamage through reduced clearance of endogenous metabolites such asoxysterols (below) and bilirubin and by reduced clearance of exogenousmetabolites such as ketoconazole.

In addition to the wider ranging impact of ketoconazole mediatedinhibition of CYP7A noted above there may be a more specific effectthrough decreased clearance of oxysterols. Oxysterols (hydroxylatedsterols) are formed as precursors to cholesterol or via subsequenthydroxylation of cholesterol. They are removed from the liver viaconversion to bile acids or solubilized in the bile. The most abundanthuman enzyme able to initiate the conversion of oxysterols to bile acidsis CYP7A (Norlin et al. (2000). “Oxysterol 7 alpha-hydroxylase activityby cholesterol 7 alpha-hydroxylase (CYP7A).” J Biol Chem 275(44):34046-53), and, as noted above, ketoconazole can inhibit this enzyme aswell as increase the levels of some oxysterols (Miettinen 1988, supra).If the conversion fails or bile flow falls, oxysterols can accumulateand liver damage may occur. Oxysterols are cytotoxic to a variety ofcell types including hepatoma cell lines (Hietter et al. (1984).“Antagonist action of cholesterol towards the toxicity of hydroxysterolson cultured hepatoma cells.” Biochem Biophys Res Cominun 120(2): 657-64;Leighton et al. (1991). “Activation of the silent endogenouscholesterol-7-alpha-hydroxylase gene in rat hepatoma cells: a newcomplementation group having resistance to 25-hydroxycholesterol.” MolCell Biol 11(4): 2049-56; O'Callaghan et al. 1999). “Oxysterol-inducedcell death in U937 and HepG2 cells at reduced and normal serumconcentrations.” Eur J Nutr 38(6): 255-62). More specifically, one studyhas reported that H35 rat hepatoma cells die in the presence of theoxysterol 25-hydroxy cholesterol and that resistance to 25-hydroxycholesterol can be brought about by the expression of human CYP7.Ketoconazole abrogates this CYP7 mediated resistance (Leighton et al.1991, supra).

The magnitude of the decrease in bile acid synthesis and the increase inoxysterols following ketoconazole mediated CYP7A inhibition will dependon the level of CYP7B (oxysterol 7alpha hydroxylase). As CYP7B is undergenetic and physiological control (Ren et al. (2003). “Regulation ofoxysterol 7alpha-hydroxylase (CYP7B1) in the rat.” Metabolism 52(5):636-42; Jakobsson et al. (2004). “A functional C-G polymorphism in theCYP7B1 promoter region and its different distribution in Orientals andCaucasians.” Pharmacogenomics J 4(4): 245-50), it is likely that therewill be a spectrum of activities in a human population, and it could beexpected that, in some proportion of ketoconazole treated patients, thelevel of CYP7B will be insufficient to compensate for the ketoconazolemediated suppression of CYP7A. It is known that insufficient CYP7B cancause liver damage if CYP7A activity is significantly reduced. At theextreme end of this insufficiency, a complete lack of CYP7B can befatal. Thus, one study reported on the fatal liver damage that developedin an infant lacking a functional copy of CYP7B. The liver damage wassuggested to occur as a direct toxic effect as well as from inhibitionof the formation of bile acids and, possibly, from an induction ofoxidant stress. The accumulating oxysterols could not be furthermetabolized by CYP7A because this enzyme is not expressed in infants(Setchell et al. (1998). “Identification of a new inborn error in bileacid synthesis: mutation of the oxysterol 7alpha-hydroxylase gene causessevere neonatal liver disease.” J Clin Invest 102(9): 1690-703).

The observations made in human patients treated with ketoconazolerequire an explanation for why only a subset of patients develops atransient mild increase in serum liver enzymes and an even smallersubset develop a more severe reaction. It is possible that, on firstexposure to ketoconazole, CYP7A is inhibited, bile formation and flow isreduced, and oxysterols and other potentially toxic metabolites begin toaccumulate. In the majority of patients, CYP7B is expressed atsufficient levels or is induced rapidly enough that liver damage is notdetectable. It has been demonstrated that in the complete absence ofCYP7A the alternate pathway for bile acid synthesis is upregulated(Pullinger et al. 2002, supra). In this model, in approximately 1%-10%of individuals, CYP7B is expressed at lower levels and/or the inductionof CYP7B is delayed and, as a consequence, minor liver damage occurs.However CYP7B would then be upregulated, damage is limited and resolveseven in the continued exposure to ketoconazole. In a smaller number ofpatients, the induction of CYP7B may be insufficient to compensate forthe inhibition of CYP7A, and more serious liver damage occurs. Inparticularly susceptible patients, ketoconazole mediated CYP7Ainhibition could lead to ketoconazole accumulation and drugconcentrations that are high enough to initiate direct toxicities.

It is important to note that despite ketoconazole being an important,commercially available anti-fungal drug and that the hepatic reactionscaused by ketoconazole can be life threatening, there are no reports inthe literature of any evidence that directly links ketoconazole tohepatic reactions through an inhibition of CYP7A, and there are noreports in the literature that suggest the 2S,4R enantiomer would be asafer drug based on the lower IC₅₀ of this enantiomer toward CYP7A. U.S.Pat. No. 6,040,307 describes a method for determining whether a drugcould induce hepatotoxicity that utilizes hepatic microsomes derivedfrom frozen tissue. However, hepatoxicity can only be measured usingintact live hepatocytes, preferably in a live animal.

The material provided here and in Example 3 provide an internallyconsistent mechanism for the hepatic reactions caused by racemicketoconazole. Because DIO-902 has a 12 fold lower IC₅₀ toward CYP7A thandoes the 2R,4S enantiomer, patients treated with DIO-902 will have asignificantly lower incidence of hepatic reactions. The relevant drugconcentrations attained in humans, the relative levels in plasma of thetwo enantiomers, and the relative IC₅₀ values are consistent with thispossibility. The pharmacokinetic profile for the two enantiomersfollowing five BID doses of 200 mg of the racemate has been obtained.For the 2R,4S enantiomer, the IC₅₀ toward CYP7A is 0.195 microM, and ifthe intrahepatic concentration of the drug is approximately 20% of thetotal plasma drug concentration (Venkatakrishnan et al. (2000). “Effectsof the antifungal agents on oxidative drug metabolism: clinicalrelevance.” Clin Pharmacokinet 38(2): 111-80), then the enantiomer willhave to reach a total plasma concentration of approximately 1 microM(approximately 0.5 microg/mL) to inhibit effectively intrahepatic CYP7A.This is within the concentrations of this enantiomer following dosingwith 200 mg of the racemate. In contrast, DIO-902 has an IC₅₀ of 2.4microM. Thus, assuming similar drug availability, the total plasmaconcentration required for DIO-902 to inhibit CYP7A significantly wouldbe 12 microM (approximately 6.3 microg/mL). Even with the significantlygreater exposure for DIO-902, the C_(max) of this enantiomer is only 65%of this level, and thus, CYP7A is unlikely to be inhibited by DIO-902 atthese doses.

G. Clinical Study of DIO-902

A Phase 1 trial in patients with type 2 diabetes mellitus can beconducted to investigate the safety and tolerability of DIO-902. Asynopsis of such a trial is provided below. Such a trial would be thefirst human clinical study of the 2S,4R enantiomer of ketoconazoleadministered substantially free of the 2R,4S enantiomer. The primaryobjective is to evaluate the safety and tolerability of 14 daily dosesof the 2S,4R enantiomer in subjects with type 2 diabetes. The secondaryobjectives are to determine the pharmacokinetic (PK) profile in plasmaof the 2S,4R enantiomer after a single dosing and after fourteen dailydoses. In addition the pharmacodynamic activity of fourteen daily dosesof the 2S,4R enantiomer, as reflected by changes in blood pressure,cholesterol, plasma and salivary cortisol, cortisol binding globulin,measures of glycemic control (fructosamine, continuous glucosemonitoring, insulin levels, and fasting blood glucose) and plasma freefatty acids are measured.

Seven (7) dose groups are studied. Six subjects are enrolled into eachdose group. The dose groups are as follows:

Ketoconazole 400 mg po QD

2S,4R enantiomer 200 mg po QD

2S,4R enantiomer 400 mg po QD

2S,4R enantiomer 600 mg po QD

2S,4R enantiomer 800 mg po QD

2S,4R enantiomer 400 mg po BID

Placebo po QD

The dose of ketoconazole is based on the recommended maximum dose in theproduct label for use in fungal infections. Dose levels of the 2S,4Renantiomer to be studied are based on the knowledge that 50% of racemicketoconazole is the enantiomer 2S,4R, extensive clinical experience withracemic ketoconazole at doses significantly higher than thoserecommended in the drug label, toxicokinetic profiles of racemicketoconazole and the 2S,4R enantiomer in dogs, and a 28 day toxicologystudy of the 2S,4R enantiomer in dogs. The 2S,4R enantiomer and racemicketoconazole are supplied as 200 mg tablets for oral administration.Placebo tablets matching both the 2S,4R enantiomer tablets and theracemic ketoconazole tablets are also supplied.

The invention, having been described in detail and exemplified above,has a wide variety of embodiments; consequently, while certainembodiments of the invention have been described herein in detail,numerous alternative embodiments are contemplated as falling within thescope of the following claims.

All publications and patent documents (patents, published patentapplications, and unpublished patent applications) cited herein areincorporated herein by reference as if each such publication or documentwas specifically and individually indicated to be incorporated herein byreference. Citation of publications and patent documents is not intendedas an admission that any such document is pertinent prior art, nor doesit constitute any admission as to the contents or date of the same.

What is claimed is:
 1. A method for treating a disease or conditionassociated with elevated cortisol levels in a patient in need of suchtreatment, said method comprising administering a composition comprisinga ketoconazole component to said patient, wherein at least 90% of theketoconazole component is comprised of the 2S,4R ketoconazoleenantiomer.
 2. A method for treating a disease or condition associatedwith elevated cortisol levels in a patient in need of such treatment,said method comprising administering a composition comprising aketoconazole component to said patient, wherein at least 95% of theketoconazole component is comprised of the 2S,4R ketoconazoleenantiomer.
 3. A method for treating a disease or condition associatedwith elevated cortisol levels in a patient in need of such treatment,said method comprising administering a composition comprising aketoconazole component to said patient, wherein at least 99% of theketoconazole component is comprised of the 2S,4R ketoconazoleenantiomer.